Methods to improve vector expression and genetic stability

ABSTRACT

The present invention relates to methods of developing gene inserts that are more compatible with the host vectors by modifying a protein sequence to lessen potential interference with vector propagation while ensuring that the protein is expressed and processed efficiently and maintains desired structural features, and designing a gene with a nucleotide sequence that resembles the base composition of the host vector genome.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a divisional of U.S. application Ser. No. 13/792,103filed Mar. 10, 2013, which claims priority to U.S. provisional patentapplication Ser. No. 61/617,368 filed Mar. 29, 2012. Reference is madeto U.S. patent application Ser. No. 12/708,940 filed Feb. 19, 2010 andU.S. provisional patent application Serial Nos. 61/537,497 filed Sep.21, 2011; 61/552,240 filed Oct. 27, 2011 and 61/614,584 filed Mar. 23,2012.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FEDERAL FUNDING LEGEND

This invention was supported, in part, by CAVD Grant ID: 38606, CAVDGrant ID: OPP1033117 and NIAID R01: 1R01A1084840-01. The federalgovernment may have certain rights to this invention.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 20, 2013, isnamed 43094.01.2021_SL.txt and is 14,631 bytes in size.

FIELD OF THE INVENTION

The present invention relates to methods for tailoring gene insertdesigns to improve protein expression, vector propagation, and geneticstability in individual vector platforms

BACKGROUND OF THE INVENTION

Problems encountered frequently during vaccine delivery vectordevelopment include poor foreign protein expression, inefficient orincomplete post-translational processing of the immunogen, diminishedvector propagation, and gene insert instability. These problems areoften related to the foreign gene being nonessential for vectorpropagation and the negative effect on replicative fitness that often isconferred by the biological or physical characteristics of thenucleotide sequence or the encoded protein.

Earlier ‘gene optimization’ procedures used to develop gene inserts forvaccine vectors focused primarily on designing synthetic codingsequences with the characteristics of highly expressed cellular mRNAs(Andre et al. 1998. J Virol 72:1497-1503, Barouch 2006. The Journal ofpathology 208:283-289, Donnelly et al. 1997. DNA vaccines. Annu RevImmunol 15:617-648 and Haas et al. 1996. Codon usage limitation in theexpression of HIV-1 envelope glycoprotein. Current biology: CB6:315-324). Although this general optimization approach often increasesexpression of the encoded polypeptide, it also can result in a geneinsert that is poorly compatible with the vector because the expressedprotein is cytotoxic and/or the engineered nucleotide sequence isdifficult to replicate and unstable. Accordingly, there is a need todevelop a gene design approach that makes it possible to abundantlyexpress foreign proteins while also reducing the negative effect causedby introducing foreign gene sequences into a vector genetic background.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention relates to methods for tailoring gene insertdesigns to improve protein expression, vector propagation, and geneticstability in individual vector platforms. Earlier gene ‘optimization’procedures were primarily directed to improving expression inprokaryotic or eukaryotic cell substrates and organisms. The methoddescribed herein may be utilized to prepare foreign gene inserts that 1)closely resemble the nucleotide composition of the vector geneticbackground; 2) lack sequences that might cause insert instability duringvector propagation; and, 3) lack sequences known to inhibit expressionin eukaryotic cells.

To reduce the negative selective pressure caused by insertion of aforeign gene sequence while also maintaining abundant protein expressionneeded to develop an immunogenic vaccine, Applicants have developed anew strategy for designing protein-coding sequences for use in vaccinevectors. In summary, the objective of the method is to develop a geneinsert that is more compatible with the host vector by 1) modifying theprotein sequence to lessen potential interference with vectorpropagation while ensuring that the polypeptide is expressed andprocessed efficiently and maintains desired structural features, and 2)designing the gene with a nucleotide sequence that resembles the basecomposition of the host vector genome.

The objective is to develop a gene design approach that makes itpossible to abundantly express foreign proteins while also reducing thenegative effect caused by introducing foreign gene sequences into avector genetic background. The gene optimization approach incorporatesthree elements:

1. The synthetic gene is designed with a codon bias similar to thevector sequence

2. Nucleotide sequence elements in the synthetic gene that resembleknown ‘hot spots’ for mutation, inhibitors of replication or geneexpression, or have the potential to direct inappropriate RNA processingare interrupted using synonymous codons.

3. Protein functional domains that modulate translation,post-translational processing, and cellular compartmentalization may bereplaced with analogous operable elements from other polypeptides toproduce an immunogen that is expressed abundantly while having lessnegative effect on vector propagation and fitness.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 depicts antibody-binding profiles for Env immunogens expressed onthe cell surface. Env immunogens are expressed in cells transfected withplasmid vectors or infected with viral vectors and subsequently reactedwith monoclonal antibodies that react with specific domains in HIV Env.Antibody binding is detected by cell sorting.

FIG. 2 illustrates protein domain swaps used to develop hybrid Env-VSV Gproteins (EnvG) that are expressed abundantly but retain functional andantigenic properties of native Env. A) Schematic of VSV G; B) Native Envprotein; C) EnvG protein containing the VSV G signal peptide and Gcytoplasmic domain; D) Like the protein illustrated in C except that theprecise points of fusion between the Env transmembrane domain and the Gcytoplasmic domain differ and are as described by Johnson et al.(Virology 251:244-252); E) EnvG protein containing the VSV G signalpeptide, G transmembrane domain, and G cytoplasmic domain; F-H) EnvGcontaining the VSV G signal peptide, varying lengths of themembrane-proximal extracellular stem domain from G, the G transmenbranesequence, and the G cytoplasmic domain. The amino acid coordinates areprovided in Table 1.

FIGS. 3A and 3B depict a Subtype A EnvG Immunogen. Part A is a schematicshowing the protein domains incorporated into the EnvG immunogen. Part Bis the corresponding nucleotide sequence (SEQ ID NO: 4) developed forVSV vectors.

FIGS. 4A and 4B depict a Subtype C EnvG hybrid immunogen. Part Aillustrates the domains incorporated in the polypeptide. The genesequence is provided in Part B (SEQ ID NO: 5).

FIGS. 5A and 5B depict a Subtype B EnvG hybrid immunogen. Part Aillustrates the domains incorporated in the polypeptide. The genesequence is provided in Part B (SEQ ID NO: 6).

FIG. 6 depicts a Western blot analysis of EnvG proteins. Total celllysates was prepared from Vero cells infected with the VSV vectorencoding EnvG (JR-FL). The cell lysate was subjected to denaturing SDSpolyacrylamide gel electrophoresis in lane 1. VSV vector particles wereharvested from infected Vero cells and purified by centrifugationthrough a sucrose solution. 1×10⁶ or 1×10⁵ plaque-forming units weredenatured and electrophoresed in lanes 2 and 3, respectively. TheWestern blot was probed with monoclonal antibody 2F5 (Muster et al.,Journal of virology 67:6642-6647), which is specific for sequencespresent in the gp 160 precursor and the gp41 Env subunit.

FIGS. 7A and 7B depict Vero cells infected with CDV-HIV Env vectorsexpressing (A) Subtype A or (B) Subtype C Env. The plaques were stainedwith monoclonal antibody 4E10, which is specific for sequences in thegp160 precusor or the gp41 subunit of Env (Zwick et al., J Virol75:6692-6699).

FIGS. 8A-8D depict Canine distemper virus (CDV) vectors encoding theHIVCON Immunogen. The single-stranded, negative-sense, nonsegmented RNAgenome of CDV (Part A) contains 6 genes. Transcription start and stopsignals flank each gene. Three HIVCON genes have been prepared forincorporation into the CDV genome. Illustrated in Part B, a HIVCONinsert has been designed with a codon bias similar to CDV with viraltranscription start and stop signals flanking the synthetic gene. TheHIVCON coding sequence represented in Part B was modified further forinsertion downstream of the CDV P gene. The nucleotide sequence isprovided below (FIG. 9). Notably, the HIVCON coding sequence was fusedto the M (Matrix) gene of CDV to form a polycistronic coding sequence.The 2A-like element (Luke et al., J Gen Virol 89:1036-1042) insertedbetween the HIVCON and M coding sequence encodes a polypeptide cleavageelement, which allows release of the HIVCON and M polypeptides from alarger fusion protein precursor. In Part D, the strategy described inPart C was used to insert the gene downstream of CDV P except that theHIVCON coding sequence was the same as described by Letourneau et al(PLoS ONE 2:e984). The VSV signal sequence was added to all HIVCONconstructs.

FIG. 9 depicts a design of a HIVCON gene for expression from CDVvectors. An annotated nucleotide sequence (SEQ ID NO: 7) is provided forthe gene design illustrated in FIG. 8C.

FIGS. 10A and 10B depict transient expression of EnvG immunogens byplasmid DNA vectors. A) Schematic of the EnvG gene, which was expressedby the plasmid used for transient expression. B) Analysis of transfected293 cells with a panel of antibodies (Walker et al., Nature 477:466-470,Walker et al., Science 326:285-289, Zwick and Burton, Curr HIV Res5:608-624) used to probe antigenic profiles.

FIG. 11 depicts designs of Hybrid Env-F proteins. The Subtype C Env genewas further modified to generate coding sequences for hybrid Envproteins that contain domains derived from CDV F.

FIG. 12 depicts transient expression of EnvF in transfected 293T cells.Total cell lysates were prepared and analyzed by denaturing SDSpolyacrylamide gel electrophoresis. Proteins were transferred to amembrane and reacted with anti-HIV immunoglobulin. Antibody specific forbeta-actin was used as a control. M) Marker; C) untransfected controlcell lysate; E) plasmid DNA containing the native unmodified Env gene;1-4) EnvF hybrid proteins illustrated in FIG. 11.

FIG. 13 depicts four chimeric EnvF constructs (Subtype C, strain 16055)which were designed and cloned under CMV promoter in plasmid DNA vector(FIG. 11). In addition to coding sequence design described above, Fdomain substitutions were made as illustrated in FIG. 11.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for producing improved geneinserts for vaccine vectors. The strategy used for HIV immunogens may beapplied to a broad range of other polypeptides including viralglycoproteins from respiratory syncytial virus, human parainfluenzavirus, human cytomegalovirus, herpes simplex, and others.

The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer may be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

As used herein, the terms “antigen” or “immunogen” are usedinterchangeably to refer to a substance, typically a protein, which iscapable of inducing an immune response in a subject. The term alsorefers to proteins that are immunologically active in the sense thatonce administered to a subject (either directly or by administering tothe subject a nucleotide sequence or vector that encodes the protein) isable to evoke an immune response of the humoral and/or cellular typedirected against that protein.

The term “antibody” includes intact molecules as well as fragmentsthereof, such as Fab, F(ab′)₂, Fv and scFv which are capable of bindingthe epitope determinant. These antibody fragments retain some ability toselectively bind with its antigen or receptor and include, for example:

-   -   a. Fab, the fragment which contains a monovalent antigen-binding        fragment of an antibody molecule can be produced by digestion of        whole antibody with the enzyme papain to yield an intact light        chain and a portion of one heavy chain;    -   b. Fab′, the fragment of an antibody molecule can be obtained by        treating whole antibody with pepsin, followed by reduction, to        yield an intact light chain and a portion of the heavy chain;        two Fab′ fragments are obtained per antibody molecule;    -   c. F(ab′)₂, the fragment of the antibody that can be obtained by        treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   d. scFv, including a genetically engineered fragment containing        the variable region of a heavy and a light chain as a fused        single chain molecule.

General methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), which is incorporated herein byreference).

It should be understood that the proteins, including the antigens of theinvention may differ from the exact sequences illustrated and describedherein. Thus, the invention contemplates deletions, additions andsubstitutions to the sequences shown, so long as the sequences functionin accordance with the methods of the invention. In this regard,particularly preferred substitutions will generally be conservative innature, i.e., those substitutions that take place within a family ofamino acids. For example, amino acids are generally divided into fourfamilies: (1) acidic—aspartate and glutamate; (2) basic—lysine,arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar—glycine, asparagine, glutamine, cysteine, serine threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified as aromatic amino acids. It is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, or viceversa; an aspartate with a glutamate or vice versa; a threonine with aserine or vice versa; or a similar conservative replacement of an aminoacid with a structurally related amino acid, will not have a majoreffect on the biological activity. Proteins having substantially thesame amino acid sequence as the sequences illustrated and described butpossessing minor amino acid substitutions that do not substantiallyaffect the immunogenicity of the protein are, therefore, within thescope of the invention.

As used herein the terms “nucleotide sequences” and “nucleic acidsequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) sequences, including, without limitation, messenger RNA (mRNA),DNA/RNA hybrids, or synthetic nucleic acids. The nucleic acid can besingle-stranded, or partially or completely double-stranded (duplex).Duplex nucleic acids can be homoduplex or heteroduplex.

As used herein the term “transgene” may used to refer to “recombinant”nucleotide sequences that may be derived from any of the nucleotidesequences encoding the proteins of the present invention. The term“recombinant” means a nucleotide sequence that has been manipulated “byman” and which does not occur in nature, or is linked to anothernucleotide sequence or found in a different arrangement in nature. It isunderstood that manipulated “by man” means manipulated by someartificial means, including by use of machines, codon optimization,restriction enzymes, etc.

For example, in one embodiment the nucleotide sequences may be mutatedsuch that the activity of the encoded proteins in vivo is abrogated. Inanother embodiment the nucleotide sequences may be codon optimized, forexample the codons may be optimized for human use. In preferredembodiments the nucleotide sequences of the invention are both mutatedto abrogate the normal in vivo function of the encoded proteins, andcodon optimized for human use. For example, each of the Gag, Pol, Env,Nef, RT, and IN sequences of the invention may be altered in these ways.

With respect to codon optimization, the nucleic acid molecules of theinvention have a nucleotide sequence that encodes the antigens of theinvention and can be designed to employ codons that are used in thegenes of the subject in which the antigen is to be produced.Advantageously, codons are optimized using a bias that is specific forthe viral vector and not for the host cell. Many viruses, including HIVand other lentiviruses, use a large number of rare codons and, byaltering these codons to correspond to codons commonly used in thedesired subject, enhanced expression of the antigens can be achieved. Inanother embodiment, the codons used may be “humanized” codons, i.e., thecodons are those that appear frequently in highly expressed human genes(Andre et al., J. Virol. 72:1497-1503, 1998) instead of those codonsthat are frequently used by HIV. Such codon usage may provide forefficient expression of the transgenic HIV proteins in human cells. Anysuitable method of codon optimization may be used. Such methods, and theselection of such methods, are well known to those of skill in the art.In addition, there are several companies that will optimize codons ofsequences, such as Geneart (geneart.com). Thus, the nucleotide sequencesof the invention can readily be codon optimized.

The invention further encompasses nucleotide sequences encodingfunctionally and/or antigenically equivalent variants and derivatives ofthe antigens of the invention and functionally equivalent fragmentsthereof. These functionally equivalent variants, derivatives, andfragments display the ability to retain antigenic activity. Forinstance, changes in a DNA sequence that do not change the encoded aminoacid sequence, as well as those that result in conservativesubstitutions of amino acid residues, one or a few amino acid deletionsor additions, and substitution of amino acid residues by amino acidanalogs are those which will not significantly affect properties of theencoded polypeptide. Conservative amino acid substitutions areglycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;aspartic acid/glutamic acid; serine/threonine/methionine;lysine/arginine; and phenylalanine/tyrosine/tryptophan. In oneembodiment, the variants have at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% homology oridentity to the antigen, epitope, immunogen, peptide or polypeptide ofinterest.

For the purposes of the present invention, sequence identity or homologyis determined by comparing the sequences when aligned so as to maximizeoverlap and identity while minimizing sequence gaps. In particular,sequence identity may be determined using any of a number ofmathematical algorithms. A nonlimiting example of a mathematicalalgorithm used for comparison of two sequences is the algorithm ofKarlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268,modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877.

Another example of a mathematical algorithm used for comparison ofsequences is the algorithm of Myers & Miller, CABIOS 1988; 4: 11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used. Yet another useful algorithm for identifying regions oflocal sequence similarity and alignment is the FASTA algorithm asdescribed in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448.

Advantageous for use according to the present invention is the WU-BLAST(Washington University BLAST) version 2.0 software. WU-BLAST version 2.0executable programs for several UNIX platforms can be downloaded. Thisprogram is based on WU-BLAST version 1.4, which in turn is based on thepublic domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Localalignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480;Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish &States, 1993;Nature Genetics 3: 266-272; Karlin & Altschul, 1993;Proc.Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated byreference herein).

The various recombinant nucleotide sequences and antigens of theinvention are made using standard recombinant DNA and cloningtechniques. Such techniques are well known to those of skill in the art.See for example, “Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al. 1989).

The nucleotide sequences of the present invention may be inserted into“vectors.” The term “vector” is widely used and understood by those ofskill in the art, and as used herein the term “vector” is usedconsistent with its meaning to those of skill in the art. For example,the term “vector” is commonly used by those skilled in the art to referto a vehicle that allows or facilitates the transfer of nucleic acidmolecules from one environment to another or that allows or facilitatesthe manipulation of a nucleic acid molecule.

Any vector that allows expression of the antigens of the presentinvention may be used in accordance with the present invention. Incertain embodiments, the antigens and/or antibodies of the presentinvention may be used in vitro (such as using cell-free expressionsystems) and/or in cultured cells grown in vitro in order to produce theencoded antigens and/or antibodies which may then be used for variousapplications such as in the production of proteinaceous vaccines. Forsuch applications, any vector that allows expression of the antigensand/or antibodies in vitro and/or in cultured cells may be used.

For applications where it is desired that the antigens be expressed invivo, any vector that allows for the expression of the antigens of thepresent invention and is safe for use in vivo may be used. In preferredembodiments the vectors used are safe for use in humans, mammals and/orlaboratory animals.

For the antigens of the present invention to be expressed, the proteincoding sequence should be “operably linked” to regulatory or nucleicacid control sequences that direct transcription and translation of theprotein. As used herein, a coding sequence and a nucleic acid controlsequence or promoter are said to be “operably linked” when they arecovalently linked in such a way as to place the expression ortranscription and/or translation of the coding sequence under theinfluence or control of the nucleic acid control sequence. The “nucleicacid control sequence” can be any nucleic acid element, such as, but notlimited to promoters, enhancers, IRES, introns, and other elementsdescribed herein that direct the expression of a nucleic acid sequenceor coding sequence that is operably linked thereto. The term “promoter”will be used herein to refer to a group of transcriptional controlmodules that are clustered around the initiation site for RNA polymeraseII and that when operationally linked to the protein coding sequences ofthe invention lead to the expression of the encoded protein. Theexpression of the transgenes of the present invention can be under thecontrol of a constitutive promoter or of an inducible promoter, whichinitiates transcription only when exposed to some particular externalstimulus, such as, without limitation, antibiotics such as tetracycline,hormones such as ecdysone, or heavy metals. The promoter can also bespecific to a particular cell-type, tissue or organ. Many suitablepromoters and enhancers are known in the art, and any such suitablepromoter or enhancer may be used for expression of the transgenes of theinvention. For example, suitable promoters and/or enhancers can beselected from the Eukaryotic Promoter Database (EPDB).

The vectors used in accordance with the present invention shouldtypically be chosen such that they contain a suitable gene regulatoryregion, such as a promoter or enhancer, such that the antigens and/orantibodies of the invention can be expressed.

For example, when the aim is to express the antigens of the invention invitro, or in cultured cells, or in any prokaryotic or eukaryotic systemfor the purpose of producing the protein(s) encoded by that antibodyand/or antigen, then any suitable vector can be used depending on theapplication. For example, plasmids, viral vectors, bacterial vectors,protozoan vectors, insect vectors, baculovirus expression vectors, yeastvectors, mammalian cell vectors, and the like, can be used. Suitablevectors can be selected by the skilled artisan taking into considerationthe characteristics of the vector and the requirements for expressingthe antigens under the identified circumstances.

When the aim is to deliver antigens of the invention in vivo in asubject, for example in order to generate an immune response against anHIV-1 antigen and/or protective immunity against HIV-1, expressionvectors that are suitable for expression on that subject, and that aresafe for use in vivo, should be chosen. For example, in some embodimentsit may be desired to express the antigens of the invention in alaboratory animal, such as for pre-clinical testing of the HIV-1immunogenic compositions and vaccines of the invention. In otherembodiments, it will be desirable to express the antigens of theinvention in human subjects, such as in clinical trials and for actualclinical use of the immunogenic compositions and vaccine of theinvention. Any vectors that are suitable for such uses can be employed,and it is well within the capabilities of the skilled artisan to selecta suitable vector. In some embodiments it may be preferred that thevectors used for these in vivo applications are attenuated to vectorfrom amplifying in the subject. For example, if plasmid vectors areused, preferably they will lack an origin of replication that functionsin the subject so as to enhance safety for in vivo use in the subject.If viral vectors are used, preferably they are attenuated orreplication-defective in the subject, again, so as to enhance safety forin vivo use in the subject.

The present invention relates to recombinant enveloped viruses asvectors, however, other vectors may be contemplated in other embodimentsof the invention such as, but not limited to, prime boost administrationwhich may comprise administration of a recombinant envelope virus vectorin combination with another recombinant vector expressing one or moreHIV epitopes.

VSV is a practical, safe, and immunogenic vector for conducting animalstudies, and an attractive candidate for developing vaccines for use inhumans. VSV is a member of the Rhabdoviridae family of enveloped virusescontaining a nonsegmented, negative-sense RNA genome. The genome iscomposed of 5 genes arranged sequentially 3′-N-P-M-G-L-5′, each encodinga polypeptide found in mature virions. Notably, the surface glycoproteinG is a transmembrane polypeptide that is present in the viral envelopeas a homotrimer, and like Env, it mediates cell attachment andinfection.

The VSVs of U.S. Pat. Nos. 7,468,274; 7,419,829; 7,419,674; 7,344,838;7,332,316; 7,329,807; 7,323,337; 7,259,015; 7,244,818; 7,226,786;7,211,247; 7,202,079; 7,198,793; 7,198,784; 7,153,510; 7,070,994;6,969,598; 6,958,226; RE38,824; PPI5,957; 6,890,735; 6,887,377;6,867,326; 6,867,036; 6,858,205; 6,835,568; 6,830,892; 6,818,209; 56,790,641; 6,787,520; 6,743,620; 6,740,764; 6,740,635; 6,740,320;6,682,907; 6,673,784; 6,673,572; 6,669,936; 6,653,103; 6,607,912;6,558,923; 6,555,107; 6,533,855; 6,531,123; 6,506,604; 6,500,623;6,497,873; 6,489,142; 6,410,316; 6,410,313; 6,365,713; 6,348,312;6,326,487; 6,312,682; 6,303,331; 6,277,633; 6,207,455; 6,200,811;6,190,650; 6,171,862; 6,143,290; 6,133,027; 6,121,434; 6,103,462;6,069,134; 6,054,127; 6,034,073; 5,969,211; 10 5,935,822; 5,888,727;5,883,081; 5,876,727; 5,858,740; 5,843,723; 5,834,256; 5,817,491;5,792,604; 5,789,229; 5,773,003; 5,763,406; 5,760,184; 5,750,396;5,739,018; 5,698,446; 5,686,279; 5,670,354; 5,540,923; 5,512,421;5,090,194; 4,939,176; 4,738,846; 4,622,292; 4,556,556 and 4,396,628 maybe contemplated by the present invention.

The CDVs of U.S. Pat. Nos. 7,879,336; 7,833,532; 7,378,101; 7,288,265;6,673,572; 6,228,846; 5,843,456; 5,178,862 and 4,992,272 may becontemplated by the present invention.

The measles of U.S. Pat. Nos. 6,884,786; 5,578,448 and 4,016,252 may becontemplated by the present invention.

Other envelope viruses are also contemplated, such as a herpesvirus,poxvirus, hepadnavirus, flavivirus, togavirus, coronavirus, hepatitis Dvirus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus or aFilovirus.

Problems encountered frequently during vaccine delivery vectordevelopment include poor foreign protein expression, inefficient orincomplete post-translational processing of the immunogen, diminishedvector propagation, and gene insert instability. These problems areoften related to the foreign gene being nonessential for vectorpropagation and the negative effect on replicative fitness that often isconferred by the biological or physical characteristics of thenucleotide sequence or the encoded protein. To reduce the negativeselective pressure caused by insertion of a foreign gene sequence whilealso maintaining abundant protein expression needed to develop animmunogenic vaccine, Applicants have developed a new strategy fordesigning protein-coding sequences for use in vaccine vectors. Insummary, the objective of the method is to develop a gene insert that ismore compatible with the host vector by 1) modifying the proteinsequence to lessen potential interference with vector propagation whileensuring that the polypeptide is expressed and processed efficiently andmaintains desired structural features, and 2) designing the gene with anucleotide sequence that resembles the base composition of the hostvector genome.

Earlier ‘gene optimization’ procedures used to develop gene inserts forvaccine vectors focused primarily on designing synthetic codingsequences with the characteristics of highly expressed cellular mRNAs.Although this general optimization approach often increases expressionof the encoded polypeptide, it also can result in a gene insert that ispoorly compatible with the vector because the expressed protein iscytotoxic and/or the engineered nucleotide sequence is difficult toreplicate and unstable. Accordingly, Applicants' objective is to developa gene design approach that makes it possible to abundantly expressforeign proteins while also reducing the negative effect caused byintroducing foreign gene sequences into a vector genetic background.Applicants' gene optimization approach incorporates three elements:

-   -   1. The synthetic gene is designed with a codon bias similar to        the vector sequence    -   2. Nucleotide sequence elements in the synthetic gene that        resemble known ‘hot spots’ for mutation, inhibitors of        replication or gene expression, or have the potential to direct        inappropriate RNA processing are interrupted using synonymous        codons.    -   3. Protein functional domains that modulate translation,        post-translational processing, and cellular compartmentalization        can be replaced with analogous operable elements from other        polypeptides to produce an immunogen that is expressed        abundantly while having less negative effect on vector        propagation and fitness.

Below, Applicants' gene design approach is illustrated with HIV Envgenes designed specifically for expression by live vesicular stomatitisvirus (VSV) vectors. Other examples follow to show that the strategy isnot limited to VSV vectors or Env immunogens.

Development of a vaccine that will expose the immune system to aproperly configured Env immunogen that is nearly identical to thefunctional structures found on the surface of HIV particles is adifficult technical problem, because the active form of Env is amembrane-bound, unstable, multi-subunit complex. Functional Env proteinis part of trimeric glycoprotein spike that is assembled from subunitsderived from a virus-encoded precursor protein (gp160). Maturation ofgp160 involves extensive post-translational modification includingglycosylation, formation of multiple intra-chain disulfide linkages, andproteolytic cleavage into two subunits, which include the smallertransmembrane glycoprotein gp41 and the soluble extracellular subunitgp120. Subunits gp41 and gp120 are held together by noncovalentinteractions to form a monomer and three Env monomers associate into acomplex to form the functional trimeric spikes found on surface of theHIV particles and infected cells. Design and production of a vaccinethat can deliver natively configured trimeric spike immunogens must takeinto account: 1) synthesis of immunogens that are modified extensivelyand correctly; 2) assembly of a stable multisubunit complex heldtogether by labile noncovalent interactions; 3) known insolubility ofthe native gp41 subunit; and 4) the native spike complex is naturallyanchored to the lipid bilayer of cells and virus particles. Thesebiochemical features make it difficult to produce a soluble proteinimmunogen that accurately mimics a trimeric spike; thus, a vectorplatform that will direct synthesis of the native Env spike followingvaccine administration is a more practical approach.

The methods used before to improve Env expression are not applicable toall vectors and immunogen designs. For example, protein modificationsintroduced to improve expression might alter structural conformationproducing unpredictable effects on important epitopes. Nucleotidesequence optimization also can have unpredictable effects. Notably,common gene optimization procedures generally use a codon biasreflecting highly expressed mammalian mRNAs, which may not be compatiblewith viral vectors that have genomes with much different nucleotidecompositions. This can be illustrated with the HIV Env gene. Usingonline web tools to design an Env gene with a codon bias reflectinghighly expressed mammalian genes results in a coding sequence that has aguanine plus cytosine (G+C) content of approximately 60%. Gene insertswith high G+C content like this might be poorly expressed or unstablewhen inserted into vectors like those based on RNA viruses such asmeasles virus, bovine parainfluenza virus, and vesicular stomatitisvirus, which have noticeably lower genomic G+C content of 47%, 36%, and42%, respectively.

Applicants' goal is to make genetically stable vaccine vectors thatabundantly express trimeric membrane-bound Env spikes that closely mimicthe structural properties of the functional glycoprotein complex foundon HIV particles. To achieve this objective, Applicants have developed astrategy to design gene inserts that are tailored to the vector. Toensure that the modified Env immunogens encoded by Applicants' vectorsare processed accurately and retain a native configuration, Applicantshave developed a method to confirm the presence of different classes ofepitopes known to bind with virus-neutralizing antibodies (FIG. 1). Incells expressing the Env immunogens, FACS analysis is used with antibodyprobes including monoclonal antibodies that recognize the CD4 bindingsite (VRC01 and b12), determinant formed by the V1/V2 loops (PG9 andPG16), structures formed by the glycosylated sequences in the V3 loop(PGT121, PGT126, PGT128, and PGT130), the membrane-proximal externalregion (2F5 and 4E10), and unique structures formed specifically by thetrimeric spike (PGT145). The presence of epitopes is detected on thecell surface by FACs to confirm that the vector is directing expressionof a correctly processed and assembled Env spike.

The foreign gene insert optimization strategy Applicants have developedmay be illustrated using HIV Env (strain JR-FL, subtype B). Adaptationof this method to other types of gene inserts and vectors also includesEnvF fusions proteins for canine distemper virus (CDV) vectors; SIVgenes for CDV; SIV genes for VSV; HIV Gag for VSV; HIVCON for CDV.

In summary, the major elements of the Env gene insert optimizationstrategy are:

-   1. Design of the Env gene insert using a nucleotide bias    characteristic of the VSV genome-   2. Removal of RNA sequence elements that might cause VSV genome    instability, inhibit protein translation, reduce mRNA stability, or    promote unwanted RNA processing.-   3. Addition of cis-acting RNA sequences that promote efficient    translation.-   4. Design of a hybrid Env immunogen in which select Env functional    domains are substituted with sequences from the VSV glycoprotein (G)    to promote improved protein expression. Domains from G found to    improve expression while retaining Env structures recognized by    neutralizing antibodies include: a) the VSV G signal sequence    (secretory signal); b) the membrane proximal extracellular stem    domain; c) the transmembrane domain; and/or the intracellular    cytoplasmic domain. It is important to note that in some instances,    replacement of Env domains with sequences from G was not beneficial.    For example, the VSV signal sequence did not enhance expression or    genetic stability in some VSV vectors, but further investigation    revealed that substitution of the signal sequence from the cellular    CD5 protein was beneficial. Thus, the domain swap strategy is    generally applicable, but requires some empirical determination to    achieve the greatest benefit.

Nucleotide sequence design: of HIV-1 Env (subtype B, strain JR-FL) geneinserts for expression from VSV vectors includes:

-   -   1. The Optimizer Web Tool (Nucleic Acids Res 35:W126-131) was        used to generate a new Env coding sequence with a codon bias        similar to VSV.    -   2. The computer-generated sequence was then scanned for        sequences that might have a negative effect on RNA transcription        or stability, translation, or VSV genome replication. These        sequences were removed by replacing nucleotides in the synthetic        Env coding sequence with synonymous codons. Sequences targeted        for substitution included:        -   a. Most homopolymer stretches ≧5 nucleotides        -   b. Sequences resembling cellular mRNA splicing signals            identified using NNSPLICE webtool (J Comput Biol 4:311-323)        -   c. Sequences resembling known RNA instability elements (Mol            Cell Biol 15:2219-2230)        -   d. Sequences resembling the known cleavage and            polyadenylation signal (AAUAAA) (MMBR 63:405-445)        -   e. Sequences resembling transcription start and stop signal            consensus for VSV (Biochim Biophys Acta 1577:337-353)    -   3. The 5′ end of the coding sequences was modified to include a        translation initiation context resembling a consensus for the        other VSV genes (GTCATCATG)    -   4. Stop codon context was modified to resemble highly-expressed        human genes according to Kochetov (FEBS letters 440:351-355)    -   5. 5′ end includes a HindIII site for cloning into the rVSV        genomic cDNA.    -   6. For molecular cloning purposes, two sequences were edited to        remove HindIII sites in the coding sequence.    -   7. 3′ end was modified by addition of a BamHI site for cloning        into the rVSV genomic cDNA

Protein Modifications. Multiple domain swaps were tested identifyingseveral that 1) improve Env expression; 2) retain functional propertiesof native Env; and 3) preserve critical structural determinantsrecognized by neutralizing antibodies.

TABLE 1 Amino acid coordinates for sequences of HIV Env and VSV Gincorporated into EnvG hybrids illustrated in FIG. 2. N-Terminal SignalProtein Name Peptide Env (JR-FL) VSV G A VSV G 1-17 VSV G None  18-511 BEnv (JR-FL) 1-29 Env 30-847 None C EnvG-CT 1-17 VSV 30-695 483-511 DEnvG-CTR * 1-17 VSV 30-700 486-511 E EnvG-TM 1-17 VSV 30-674 463-511 FEnvG-SS 1-17 30-650 446-511 G EnvG-LS 1-17 30-650 426-511 H EnvG-XLS1-17 30-650 395-511

HIV-1 Env (Subtype A, strain BG505) Gene Insert Design for VSV. Asynthetic gene sequence encoding HIV-1 Env (subtype A, strain BG505) wasdesigned for VSV vectors as described above with some modifications.

Nucleotide sequence design:

-   -   1. The Optimizer webtool (Nucleic Acids Res 35:W126-131) was        used to generate a new BG505 Env coding sequence that has a        codon bias similar to VSV.    -   2. Homopolymer sequences >5 nucleotides (CCCCC, GGGGG) were        interrupted by substitution of at least one synonymous codon.    -   3. Homopolymer sequences >4 nculeotides (AAAA, TTTT) were        interrupted by substitution with at least one synonymous codon.    -   4. The 5′ end of the coding sequences was modified to include a        Kozak translation initiation sequence (aggaGCCACCATG (SEQ ID NO:        1)) (J Biol Chem 266:19867-19870)    -   5. An optimal translation termination signal TAAag was added to        the 3′ end of the coding sequences (FEBS letters 440:351-355)    -   6. RNA instability elements similar to UUAUUUAUU (Mol Cell Biol        15:2219-2230) were interrupted by replacing sequence with        synonymous codons.    -   7. Potential polyadenylation signals (AAUAAA) (MMBR 63:405-445)        were interrupted by substitution with synonymous codons.    -   8. Potential T7 polymerase terminators were removed: cTGAg,        gacTAAag, ctTAAac and gacTAAat to prevent inhibition of        recombinant VSV rescue from cloned DNA (Journal of molecular        biology 238:145-158)    -   9. The mRNA splice site prediction tool NNSPLICE webtool (J        Comput Biol 4:311-323) was used to predict the potential splice        sites and synonymous codons were substituted.    -   10. A NheI(GCTAGC) site was added to the 3′ end of VSV-G signal        peptide    -   11. 5′ end was modified to include a BstBI site for cloning into        the rVSV genomic cDNA.    -   12. 3′ end was modified to include a PacI site for cloning into        the rVSV genomic cDNA

A synthetic HIV Env gene (subtype B, strain JR-FL) was designed forincorporation into VSV vectors using the steps described in 2.2.1 withsome modification. The EnvG hybrid was designed specifically for makingstable VSV vectors that coexpress VSV G and Env.

-   -   1. The Optimizer webtool (Nucleic Acids Res 35:W126-131) was        used to generate a new strain JR-FL Env coding sequence that has        a codon bias similar to VSV.    -   2. Homopolymer sequences >5 nucleotides (CCCCC, GGGGG) were        interrupted by substitution of at least one synonymous codon.    -   3. Homopolymer sequences >4 nucleotides (AAAA, TTTT) were        interrupted by substitution with at least one synonymous codon.    -   4. The 5′ end of the coding sequences was modified to include a        Kozak translation initiation sequence (aggaGCCACCATG (SEQ ID NO:        1)) (J Biol Chem 266:19867-19870)    -   5. An optimal translation termination signal TGAg was added to        the 3′ end of the coding sequences (FEBS letters 440:351-355)    -   6. RNA instability elements similar to UUAUUUAUU (Mol Cell Biol        15:2219-2230) were interrupted by replacing sequence with        synonymous codons.    -   7. Potential polyadenylation signals (AAUAAA) (MMBR 63:405-445)        were interrupted by substitution with synonymous codons.    -   8. Potential T7 polymerase terminators were removed: cTGAg,        gacTAAag, ctTAAac and gacTAAat to prevent inhibition of        recombinant VSV rescue from cloned DNA (Journal of molecular        biology 238:145-158)    -   9. The mRNA splice site prediction tool NNSPLICE webtool (J        Comput Biol 4:311-323) was used to predict the potential splice        sites and synonymous codons were substituted.    -   10. 5′ end was modified to include a BstBI site for cloning into        the rVSV genomic cDNA.    -   11. 3′ end was modified to include a PacI site for cloning into        the rVSV genomic cDNA

In this example, an EnvG hybrid was designed with the cellular CD5signal peptide and the VSV G transmembrane and cytoplasmic domains.Additionally, an Alanine residue was added at the C-terminus of thesignal peptide to improve agreement with signal peptidase cleavagesignals (Nature methods 8:785-786).

It is important to note that Applicants' gene insert optimizationapproach is not restricted to VSV vectors and the strategy can bemodified for use across multiple vector platforms. Examples of Env geneinserts designed for expression from canine distemper virus (CDV)vectors are provided below. Env gene inserts with nucleotide sequenceresembling the CDV genome can be produced and functional domains fromthe CDV fusion (F) protein can be functionally substituted into Env asdescribed above for VSV G.

The gene optimization procedure also is not limited to Env immunogens.Changes to nucleotide sequence to reflect the base composition of theviral vector genome can improve the stability of other inserts. Inaddition, vector stability can be improved by altering specific proteinsignals that modulate export, posttranslational modification, orcellular localization. Examples below include an HIV gag gene insertdesigned for expression from VSV, SIV Gag and Env genes designed forexpression by CDV, and SIV Gag and Env genes designed for expressionfrom VSV, and a fusion protein called the HIVCON designed for expressionby CDV.

Finally, Applicants have found that gene insert designed as describedabove also can be used in plasmid DNA expression vectors. For example,genes designed for VSV also expressed efficiently from plasmid DNAvectors. This result indicates that elements of the gene design approachApplicants have developed have more broad applicability.

Synthetic genes encoding HIV Env were designed for expression from avector developed from an attenuated strain of canine distemper virus(CDV). Vectors expressing HIV Env subtype A (BG505) and subtype C(16055) Env have been isolated.

A synthetic HIV Env gene (subtype A, BG505) was designed forincorporation into CDV vectors steps similar to those described abovewith modifications. Importantly, the synthetic gene was designed using acodon bias characteristic of the CDV genome.

The gene was designed using the following steps:

-   -   1. The Optimizer Web Tool (Nucleic Acids Res 35:W126-131) was        used to generate a synthetic Env gene with a nucleotide content        similar to the CDV genome. CDV codon bias was retrieved from        database (25).    -   2. Homopolymer sequences >5 nucleotides were interrupted by        substitution of at least one synonymous codon.    -   3. The 5′ end of the coding sequences was modified to include a        Kozak translation initiation sequence (aggaGCCACCATG (SEQ ID NO:        1)) (J Biol Chem 266:19867-19870)    -   4. RNA instability elements similar to UUAUUUAUU (Mol Cell Biol        15:2219-2230) were interrupted by replacing sequence with        synonymous codons.    -   5. Potential polyadenylation signals (AAUAAA) (MMBR 63:405-445)        were interrupted by substitution with synonymous codons.    -   6. Potential T7 polymerase terminators were removed to prevent        inhibition of recombinant CDV rescue from cloned DNA (Journal of        molecular biology 238:145-158).    -   7. The mRNA splice site prediction webtool NNSPLICE (J Comput        Biol 4:311-323) was used to predict the potential splice sites        and synonymous codons were substituted.    -   8. Adjust insert length to ensure that introduction into the CDV        genome would follow the ‘Rule-of-Six” (i.e. total CDV genome        length is multiples of 6) (J Virol 72:891-899).

Protein domain modifications: In this example, the Env genes weredesigned to encode a truncated Env proteins that lack the cytoplasmictail. Nucleotide sequence design: An SIV (strain SIVmac239) Env gene wasdesigned for expression by VSV vectors. As described below:

-   -   1. Replace the SIV signal sequence with VSV signal sequence,        keeping the first amino acid after cleavage on SIV Env.    -   2. Replace the SIV Env cytoplasmic tail with VSV G.    -   3. The Optimizer Web Tool (Nucleic Acids Res 35:W126-131) was        used to generate synthetic coding sequence with a codon bias        similar to VSV based on the codon usage for Vesicular Stomatitis        Indiana Virus (Nucleic Acids Res 28:292).    -   4. Most homopolymer stretches A nucleotides were interrupted by        substitution of one or more synonymous codon(s), using the VSV        codon usage table to maintain codon bias similar to VSV.    -   5. The 5′ end of the coding sequences was modified to include a        Kozak translation initiation sequence (aggaGCCACCATG(SEQ ID NO:        1)) (J Biol Chem 266:19867-19870)    -   6. An optimal translation termination signal TAAag was added to        the 3′ end of the coding sequences (FEBS letters 440:351-355)    -   7. The mRNA splice site prediction webtool NNSPLICE (J Comput        Biol 4:311-323) was used to predict the potential splice sites,        which were substituted with synonymous codons.    -   8. RNA instability elements similar to UUAUUUAUU (Mol Cell Biol        15:2219-2230) were interrupted by replacing sequence with        synonymous codons.    -   9. Potential polyadenylation signals (AAUAAA) (MMBR 63:405-445)        were interrupted by substitution with synonymous codons.    -   10. Potential T7 polymerase terminators were removed: cTGAg,        gacTAAag, ctTAAac and gacTAAat to prevent inhibition of        recombinant VSV rescue from cloned DNA (21)    -   11. 5′ end includes a BstBI site and the 3′ end includes a PacI        site for cloning into the rVSV genomic cDNA in place of VSV G.

The C-terminal cytoplasmic domain was removed from SIV Env.

The HIVCON is an HIV immunogen designed by Letourneau et al (PLoS ONE2:e984). The immunogen is a fusion protein composed of relatively short,highly conserved sequence elements from the HIV proteome, which isintended to elicit cellular immunity against targets that cannot beeasily mutated by the virus. The HIVCON insert designed by Hanke et alwas designed specifically for expression by plasmid DNA and DNA virusvectors. Applicants have made several new gene inserts designed forincorporation into canine distemper virus (CDV) vectors, which have asmall negative-sense, single-stranded RNA genome.

To design a gene that was more compatible with a CDV vector, a syntheticHIVCON coding sequence (corresponding to FIG. 8C) was prepared using thefollowing steps:

-   1. The optimizer Web Tool (Nucleic Acids Res 35:W126-131) was used    to generate an HIVCON coding sequence that has a codon bias similar    to CDV based on the Codon Usage for Canine Distemper Virus (Nucleic    Acids Res 28:292).-   2. All homopolymer stretches ≧4-5 nucleotides were interrupted using    an alternative codon from the CDV codon usage table to maintain    codon bias similar to CDV.-   3. All sequences that resemble morbillivirus transcription stop or    start signals (J Virol 75:921-933, Virology 193:66-72) were    interrupted using an alternative codon from the CDV codon usage    table.-   4. Potential RNA splicing signals were identified using the webtool    NNSPLICE (J Comput Biol 4:311-323) and subsequently interrupted by    substitution with synonymous codons-   5. RNA instability elements similar to 5-UUAUUUAUU-3′ (Mol Cell Biol    15:2219-2230) were interrupted by silent mutation using an    alternative codon from the CDV codon usage table to maintain codon    bias similar to CDV.-   6. Two sequences resembling T7 RNA polymerase terminators were    interrupted using synonymous codons (Journal of molecular biology    238:145-158).-   7. A Kozak sequence GGAGCCACC was inserted to improve translation    (Gene 234:187-208).-   8. To increase the stability of the vector and the integrity of the    insert, a 2A-like peptide motif from Thosea asigna virus    (EGRGSLLTCGDVEENPGP(SEQ ID NO: 2)) (J Gen Virol 89:1036-1042) was    added to fuse HIVCON coding sequence with the with CDV (M) gene.-   9. Coding sequence for the VSV G leader (MKCLLYLAFLFIGVNCK (SEQ ID    NO: 3)) was also incorporated at the N-terminus to promote secretion    of the polypeptide immunogen.

Protein modifications: The VSV signal peptide was added to theN-terminus. At the C-terminus, a 2A-like polypeptide cleavage signal (JGen Virol 89:1036-1042) was added between the HIVCON and matrix codingsequences. This creates a polycistronic HIVCON-Matrix gene that encodestwo polypeptides as depicted in FIG. 9.

To demonstrate that genes designed by Applicants' approach were notrestricted to use in RNA virus vectors like CDV and VSV, Applicantsdeveloped plasmid vectors encoding the hybrid the EnvG protein describedearlier (Clade A). In addition, using the basic approach describedabove, Applicants also designed hybrid Env proteins (EnvF) in whichdomains of Env were replaced with sequences from the CDV fusion (F)protein. EnvG and EnvF genes were cloned into plasmid DNA vectors underthe control of the under the control of the cytomegalovirus promoter andenhancer. Flow cytometry (EnvG) or Western blotting (EnvF) was used toevaluate transient expression of Env protein.

Plasmid DNA containing the EnvG (subtype A) described above wastransfected into 293T cells. Cell surface expression of the EnvGproteins and its antigenic profile are illustrated in the cell sortinganalysis shown in FIG. 10.

Western blot analysis demonstrated that all four constructs wereexpressed from transfected plasmids (FIG. 12). The four EnvF fusionprotein also were analyzed to determine whether they retained fusionfunction of the natural HIV Env protein. EnvF2 and EnvF4 were positivefor cell membrane fusion indicating that the domain substitutions didnot abolish this function (data not shown). All four EnvF proteins werepositive for binding to monoclonal antibodies PG9, PG16, B6, B12, andVRC01 when expressed on the cell surface following transient expression.

The nucleotide sequences and vectors of the invention can be deliveredto cells, for example if the aim is generate a viral particlescontaining the desired antigenic protein. Suitable transfection,transformation, or gene delivery methods can be used as part of thisobjective. Such methods are well known by those skilled in the art, andone of skill in the art would readily be able to select a suitablemethod depending on the nature of the nucleotide sequences, vectors, andcell types used. For example, transfection, transformation,microinjection, infection, electroporation, lipofection, orliposome-mediated delivery could be used. Generation of the viralparticles containing the desired antigens can be carried out in anysuitable type of host cells, such as bacterial cells, yeast, insectcells, and mammalian cells. The antigens of the invention can also beexpressed including using in vitro transcription/translation systems.All of such methods are well known by those skilled in the art, and oneof skill in the art would readily be able to select a suitable methoddepending on the nature of the nucleotide sequences, vectors, and celltypes used.

The viral particles may be treated with formalin, glutaraldehyde, orother chemicals that limit viral replication. The viral particles may betreated with physical methods such as radiation, heat or adsorption ontosurfaces with the result that viral replication is impaired. A virusinactivation regimen that preserves maximal antigenicity of Env isdesirable. For example, VSV particles containing EnvG described inherein, can be used to make an inactivated VSV (iVSV-Env) particle foruse as a vaccine immunogen. The inactivation kinetics of three knownvirus inactivating agents, including formalin, beta propiolactone (BPL),and ultraviolet (UV) light may be determined. Inactivation may beassessed by measuring plaque-forming units (pfu) over time.

Antigenicity of the Env spike may be assessed using a panel ofwell-characterized neutralizing antibodies directed at sites ofvulnerability on the HIV Env spike including but not necessarily limitedto the: CD4 binding site (CD4bs), mper epitopes recognized by 2F5 and4E10 bNAbs, V1/V2 quaternary epitopes (QNE) recognized by PG9, PG16,CHO1 and Glycan-binding bNAbs recognized by PGT bNAbs (PGT121, PGT125,PGT128, PGT130).

The live or inactivated viral particles may be combined with adjuvantsincluding, but not limited to, aluminum phosphate, aluminum hydroxide,iscoms (e.g. IscoMatrix), monphosphoryl lipid A, CpG-containingoligonucleotides, Adjuplex, cytokines (e.g. IL12), alone or incombination. The immunogenicity of an adjuvanted live or iVSV-Env forthe induction of functional immune responses in experimental animals maybe determined.

The optimal dose and immunization regimen for induction of Env-specificantibody response using adjuvants including aluminum phosphate andISCOMATRIX® may be determined in animals, such as rabbits. The inductionof neutralizing antibody responses using a panel of tier 1 and tier 2viruses representing clades A, B and C may be assessed. In non-humanprimates, dose-ranging studies may be performed to determine the optimalvaccination regimen for iVSV chimeras. ELISA and competition-bindingassays may be utilized to determine the breadth of response to the 4major sites of vulnerability on HIV. The induction of neutralizingantibody responses may be assessed by using a panel of tier 1 and tier 2viruses representing clades A, B and C.

Pre-GMP cell lines and virus seed stocks may be produced for generationof vaccines for clinical trials. Furthermore, pre-GMP Vero cell linethat stably expresses CD4 and CCR5 may be developed. Pre-master seedstocks of VSV chimeras expressing functional Glade A, B and C envelopetrimers may be developed and characterized.

In preferred embodiments, the nucleotide sequences, antigens of theinvention are administered in vivo, for example where the aim is toproduce an immunogenic response in a subject. A “subject” in the contextof the present invention may be any animal. For example, in someembodiments it may be desired to express the transgenes of the inventionin a laboratory animal, such as for pre-clinical testing of the HIV-1immunogenic compositions and vaccines of the invention. In otherembodiments, it will be desirable to express the antigens of theinvention in human subjects, such as in clinical trials and for actualclinical use of the immunogenic compositions and vaccine of theinvention. In preferred embodiments the subject is a human, for examplea human that is infected with, or is at risk of infection with, HIV-1.

For such in vivo applications the nucleotide sequences, antigens of theinvention are preferably administered as a component of an immunogeniccomposition which may comprise the nucleotide sequences and/or antigensof the invention in admixture with a pharmaceutically acceptablecarrier. The immunogenic compositions of the invention are useful tostimulate an immune response against HIV-1 and may be used as one ormore components of a prophylactic or therapeutic vaccine against HIV-1for the prevention, amelioration or treatment of AIDS.

The compositions of the invention may be injectable suspensions,solutions, sprays, lyophilized powders, syrups, elixirs and the like.Any suitable form of composition may be used. To prepare such acomposition, having the desired degree of purity, is mixed with one ormore pharmaceutically acceptable carriers and/or excipients. Thecarriers and excipients must be “acceptable” in the sense of beingcompatible with the other ingredients of the composition. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include, but are not limitedto, water, saline, phosphate buffered saline, dextrose, glycerol,ethanol, or combinations thereof, buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN′, PLURONICS' orpolyethylene glycol (PEG).

An immunogenic or immunological composition can also be formulated inthe form of an oil-in-water emulsion. The oil-in-water emulsion can bebased, for example, on light liquid paraffin oil (European Pharmacopeatype); isoprenoid oil such as squalane, squalene, EICOSANE™ ortetratetracontane; oil resulting from the oligomerization of alkene(s),e.g., isobutene or decene; esters of acids or of alcohols containing alinear alkyl group, such as plant oils, ethyl oleate, propylene glycoldi(caprylate/capra^(te)), glyceryl ^(t)ri(caprylate/caprate) orpropylene glycol dioleate; esters of branched fatty acids or alcohols,e.g., isostearic acid esters. The oil advantageously is used incombination with emulsifiers to form the emulsion. The emulsifiers canbe nonionic surfactants, such as esters of sorbitan, mannide—(e.g.,anhydromannitol oleate), glycerol, polyglycerol, propylene glycol, andoleic, isostearic, ricinoleic, or hydroxystearic acid, which areoptionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymerblocks, such as the Pluronic® products, e.g., L121. The adjuvant can bea mixture of emulsifier(s), micelle-forming agent, and oil such as thatwhich is commercially available under the name Provax® (IDECPharmaceuticals, San Diego, Calif.).

The immunogenic compositions of the invention can contain additionalsubstances, such as wetting or emulsifying agents, buffering agents, oradjuvants to enhance the effectiveness of the vaccines (Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.)1980).

Adjuvants may also be included. Adjuvants include, but are not limitedto, mineral salts (e.g., A1K(SO₄)₂, AlNa(SO₄)₂, AlNH(SO₄)₂, silica,alum, Al(OH)₃, Ca3(PO₄)₂, kaolin, or carbon), polynucleotides with orwithout immune stimulating complexes (ISCOMs) (e.g., CpGoligonucleotides, such as those described in Chuang, T. H. et al, (2002)J. Leuk. Biol. 71(3): 538-44; Ahmad-Nejad, P. et al (2002) Eur. J.Immunol. 32(7): 1958-68; poly IC or poly AU acids, polyarginine with orwithout CpG (also known in the art as IC31; see Schellack, C. et al(2003) Proceedings of the 34th Annual Meeting of the German Society ofImmunology; Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508),JuvaVax™ (U.S. Pat. No. 6,693,086), certain natural substances (e.g.,wax D from Mycobacterium tuberculosis, substances found inCornyebacterium parvum, Bordetella pertussis, or members of the genusBrucella), flagellin (Toll-like receptor 5 ligand; see McSorley, S. J.et al (2002) J. Immunol. 169(7): 3914-9), saponins such as QS21, QS17,and QS7 (U.S. Pat. Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495),monophosphoryl lipid A, in particular, 3-de-O-acylated monophosphoryllipid A (3D-MPL), imiquimod (also known in the art as IQM andcommercially available as Aldara®; U.S. Pat. Nos. 4,689,338; 5,238,944;Zuber, A. K. et al (2004) 22(13-14): 1791-8), and the CCR5 inhibitorCMPD167 (see Veazey, R. S. et al (2003) J. Exp. Med. 198: 1551-1562).

Aluminum hydroxide or phosphate (alum) are commonly used at 0.05 to 0.1%solution in phosphate buffered saline. Other adjuvants that can be used,especially with DNA vaccines, are cholera toxin, especiallyCTA1-DD/ISCOMs (see Mowat, A. M. et al (2001) J. Immunol. 167(6):3398-405), polyphosphazenes (Allcock, H. R. (1998) App. OrganometallicChem. 12(10-11): 659-666; Payne, L. G. et al (1995) Pharm. Biotechnol.6: 473-93), cytokines such as, but not limited to, IL-2, IL-4, GM-CSF,IL-12, IL-15 IGF-1, IFN-α, IFN-β, and IFN-γ (Boyer et al., (2002) J.Liposome Res. 121:137-142; WO01/095919), immunoregulatory proteins suchas CD40L (ADX40; see, for example, WO03/063899), and the CD1a ligand ofnatural killer cells (also known as CRONY or a-galactosyl ceramide; seeGreen, T. D. et al, (2003) J. Virol. 77(3): 2046-2055),immunostimulatory fusion proteins such as IL-2 fused to the Fc fragmentof immunoglobulins (Barouch et al., Science 290:486-492, 2000) andco-stimulatory molecules B7.1 and B7.2 (Boyer), all of which can beadministered either as proteins or in the form of DNA, on the sameexpression vectors as those encoding the antigens of the invention or onseparate expression vectors.

In an advantageous embodiment, the adjuvants may be lecithin is combinedwith an acrylic polymer (Adjuplex-LAP), lecithin coated oil droplets inan oil-in-water emulsion (Adjuplex-LE) or lecithin and acrylic polymerin an oil-in-water emulsion (Adjuplex-LAO) (Advanced BioAdjuvants(ABA)).

The immunogenic compositions can be designed to introduce the nucleicacids or expression vectors to a desired site of action and release itat an appropriate and controllable rate. Methods of preparingcontrolled-release formulations are known in the art. For example,controlled release preparations can be produced by the use of polymersto complex or absorb the immunogen and/or immunogenic composition. Acontrolled-release formulation can be prepared using appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) known to provide thedesired controlled release characteristics or release profile. Anotherpossible method to control the duration of action by acontrolled-release preparation is to incorporate the active ingredientsinto particles of a polymeric material such as, for example, polyesters,polyamino acids, hydrogels, polylactic acid, polyglycolic acid,copolymers of these acids, or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these active ingredients intopolymeric particles, it is possible to entrap these materials intomicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacrylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed in NewTrends and Developments in Vaccines, Voller et al. (eds.), UniversityPark Press, Baltimore, Md., 1978 and Remington's PharmaceuticalSciences, 16th edition.

Suitable dosages of the nucleic acids and expression vectors of theinvention (collectively, the immunogens) in the immunogenic compositionof the invention can be readily determined by those of skill in the art.For example, the dosage of the immunogens can vary depending on theroute of administration and the size of the subject. Suitable doses canbe determined by those of skill in the art, for example by measuring theimmune response of a subject, such as a laboratory animal, usingconventional immunological techniques, and adjusting the dosages asappropriate. Such techniques for measuring the immune response of thesubject include but are not limited to, chromium release assays,tetramer binding assays, IFN-γ ELISPOT assays, IL-2 ELISPOT assays,intracellular cytokine assays, and other immunological detection assays,e.g., as detailed in the text “Antibodies: A Laboratory Manual” by EdHarlow and David Lane.

When provided prophylactically, the immunogenic compositions of theinvention are ideally administered to a subject in advance of infection,such as HIV infection, or evidence of HIV infection, or in advance ofany symptom due to AIDS, especially in high-risk subjects. Theprophylactic administration of the immunogenic compositions can serve toprovide protective immunity of a subject against HIV-1 infection or toprevent or attenuate the progression of AIDS in a subject alreadyinfected with HIV-1. When provided therapeutically, the immunogeniccompositions can serve to ameliorate and treat AIDS symptoms and areadvantageously used as soon after infection as possible, preferablybefore appearance of any symptoms of AIDS but may also be used at (orafter) the onset of the disease symptoms.

The immunogenic compositions can be administered using any suitabledelivery method including, but not limited to, intramuscular,intravenous, intradermal, mucosal, and topical delivery. Such techniquesare well known to those of skill in the art. More specific examples ofdelivery methods are intramuscular injection, intradermal injection, andsubcutaneous injection. However, delivery need not be limited toinjection methods. Further, delivery of DNA to animal tissue has beenachieved by cationic liposomes (Watanabe et al., (1994) Mol. Reprod.Dev. 38:268-274; and WO 96/20013), direct injection of naked DNA intoanimal muscle tissue (Robinson et al., (1993) Vaccine 11:957-960;Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994)Virology 199: 132-140; Webster et al., (1994) Vaccine 12: 1495-1498;Davis et al., (1994) Vaccine 12: 1503-1509; and Davis et al., (1993)Hum. Mol. Gen. 2: 1847-1851), or intradermal injection of DNA using“gene gun” technology (Johnston et al., (1994) Meth. Cell Biol.43:353-365). Alternatively, delivery routes can be oral, intranasal orby any other suitable route. Delivery may also be accomplished via amucosal surface such as the anal, vaginal or oral mucosa. Immunizationschedules (or regimens) are well known for animals (including humans)and can be readily determined for the particular subject and immunogeniccomposition. Hence, the immunogens can be administered one or more timesto the subject. Preferably, there is a set time interval betweenseparate administrations of the immunogenic composition. While thisinterval varies for every subject, typically it ranges from 10 days toseveral weeks, and is often 2, 4, 6 or 8 weeks. For humans, the intervalis typically from 2 to 6 weeks. The immunization regimes typically havefrom 1 to 6 administrations of the immunogenic composition, but may haveas few as one or two or four. The methods of inducing an immune responsecan also include administration of an adjuvant with the immunogens. Insome instances, annual, biannual or other long interval (5-10 years)booster immunization can supplement the initial immunization protocol.

The present methods also include a variety of prime-boost regimens, forexample DNA prime-VSV boost regimens. In these methods, one or morepriming immunizations are followed by one or more boostingimmunizations. The actual immunogenic composition can be the same ordifferent for each immunization and the type of immunogenic composition(e.g., containing protein or expression vector), the route, andformulation of the immunogens can also be varied. For example, if anexpression vector is used for the priming and boosting steps, it caneither be of the same or different type (e.g., DNA or bacterial or viralexpression vector). One useful prime-boost regimen provides for twopriming immunizations, four weeks apart, followed by two boostingimmunizations at 4 and 8 weeks after the last priming immunization. Itshould also be readily apparent to one of skill in the art that thereare several permutations and combinations that are encompassed using theDNA, bacterial and viral expression vectors of the invention to providepriming and boosting regimens.

The prime-boost regimen can also include VSV vectors that derive their Gprotein or G/Stem protein from different serotype vesicular stomatitisviruses (Rose N F, Roberts A, Buonocore L, Rose J K. Glycoproteinexchange vectors based on vesicular stomatitis virus allow effectiveboosting and generation of neutralizing antibodies to a primary isolateof human immunodeficiency virus type 1. J Virol. 2000December;74(23):10903-10). The VSV vectors used in these examplescontain a G or G/Stem protein derived from the Indiana serotype of VSV.Vectors can also be constructed to express G or G/Stem molecules derivedfrom other VSV serotypes (i.e. vesicular stomatitis New Jersey virus orvesicular stomatitis Alagoas virus) or other vesiculoviruses (i.e.Chandipura virus, Cocal virus, Isfahan virus). Thus a prime may bedelivered in the context of a G or G/Stem moelcule that is from theIndiana serotype and the immune system can be boosted with a vector thatexpresses epitopes in the context of second serotype like New Jersey.This circumvents anti-G immunity elicited by the prime, and helps focusthe boost response against the foreign epitope.

A specific embodiment of the invention provides methods of inducing animmune response against HIV in a subject by administering an immunogeniccomposition of the invention, preferably which may comprise an VSVvector containing RNA encoding one or more of the epitopes of theinvention, one or more times to a subject wherein the epitopes areexpressed at a level sufficient to induce a specific immune response inthe subject. Such immunizations can be repeated multiple times at timeintervals of at least 2, 4 or 6 weeks (or more) in accordance with adesired immunization regime.

The immunogenic compositions of the invention can be administered alone,or can be co-administered, or sequentially administered, with other HIVimmunogens and/or HIV immunogenic compositions, e.g., with “other”immunological, antigenic or vaccine or therapeutic compositions therebyproviding multivalent or “cocktail” or combination compositions of theinvention and methods of employing them. Again, the ingredients andmanner (sequential or co-administration) of administration, as well asdosages can be determined taking into consideration such factors as theage, sex, weight, species and condition of the particular subject, andthe route of administration.

When used in combination, the other HIV immunogens can be administeredat the same time or at different times as part of an overallimmunization regime, e.g., as part of a prime-boost regimen or otherimmunization protocol. In an advantageous embodiment, the other HIVimmunogen is env, preferably the HIV env trimer.

Many other HIV immunogens are known in the art, one such preferredimmunogen is HIVA (described in WO 01/47955), which can be administeredas a protein, on a plasmid (e.g., pTHr.HIVA) or in a viral vector (e.g.,MVA.HIVA). Another such HIV immunogen is RENTA (described inPCT/US2004/037699), which can also be administered as a protein, on aplasmid (e.g., pTHr.RENTA) or in a viral vector (e.g., MVA.RENTA).

For example, one method of inducing an immune response against HIV in ahuman subject may comprise administering at least one priming dose of anHIV immunogen and at least one boosting dose of an HIV immunogen,wherein the immunogen in each dose can be the same or different,provided that at least one of the immunogens is an epitope of thepresent invention, a nucleic acid encoding an epitope of the inventionor an expression vector, preferably a VSV vector, encoding an epitope ofthe invention, and wherein the immunogens are administered in an amountor expressed at a level sufficient to induce an HIV-specific immuneresponse in the subject. The HIV-specific immune response can include anHIV-specific T-cell immune response or an HIV-specific B-cell immuneresponse. Such immunizations can be done at intervals, preferably of atleast 2-6 or more weeks.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1 Introduction

Problems encountered frequently during vaccine delivery vectordevelopment include poor foreign protein expression, inefficient orincomplete post-translational processing of the immunogen, diminishedvector propagation, and gene insert instability. These problems areoften related to the foreign gene being nonessential for vectorpropagation and the negative effect on replicative fitness that often isconferred by the biological or physical characteristics of thenucleotide sequence or the encoded protein. To reduce the negativeselective pressure caused by insertion of a foreign gene sequence whilealso maintaining abundant protein expression needed to develop animmunogenic vaccine, Applicants have developed a new strategy fordesigning protein-coding sequences for use in vaccine vectors. Insummary, the objective of the method is to develop a gene insert that ismore compatible with the host vector by 1) modifying the proteinsequence to lessen potential interference with vector propagation whileensuring that the polypeptide is expressed and processed efficiently andmaintains desired structural features, and 2) designing the gene with anucleotide sequence that resembles the base composition of the hostvector genome.

Earlier ‘gene optimization’ procedures used to develop gene inserts forvaccine vectors focused primarily on designing synthetic codingsequences with the characteristics of highly expressed cellular mRNAs(1, 2, 5, 10). Although this general optimization approach oftenincreases expression of the encoded polypeptide, it also can result in agene insert that is poorly compatible with the vector because theexpressed protein is cytotoxic and/or the engineered nucleotide sequenceis difficult to replicate and unstable. Accordingly, Applicants'objective is to develop a gene design approach that makes it possible toabundantly express foreign proteins while also reducing the negativeeffect caused by introducing foreign gene sequences into a vectorgenetic background. Applicants' gene optimization approach incorporatesthree elements:

-   -   4. The synthetic gene is designed with a codon bias similar to        the vector sequence    -   5. Nucleotide sequence elements in the synthetic gene that        resemble known ‘hot spots’ for mutation, inhibitors of        replication or gene expression, or have the potential to direct        inappropriate RNA processing are interrupted using synonymous        codons.    -   6. Protein functional domains that modulate translation,        post-translational processing, and cellular compartmentalization        can be replaced with analogous operable elements from other        polypeptides to produce an immunogen that is expressed        abundantly while having less negative effect on vector        propagation and fitness.

Below, Applicants' gene design approach is illustrated with HIV Envgenes designed specifically for expression by live vesicular stomatitisvirus (VSV) vectors. Other examples follow to show that the strategy isnot limited to VSV vectors or Env immunogens.

Example 2 Env Gene Inserts Designed for VSV Vectors

Objective of Env Immunogen Design.

An HIV vaccine that elicits durable humoral immunity, which includesantibodies that neutralize a broad spectrum of HIV isolates, willdecrease the rate of infection significantly. Recent studies have shownthat potent virus-neutralizing antibodies active against a wide range ofHIV strains do emerge in about 10-20% of infected patients clearlydemonstrating that the human immune system can produce the requisiteimmunoglobulins (6, 19, 31, 34). These HIV-neutralizing antibodies arespecific for the viral envelope glycoprotein (Env), which is the viralprotein that directs cell attachment and entry.

Antibodies that will neutralize a significant range of HIV strains havebeen elicited only in response to HIV infection (6, 19, 31, 34). Incontrast, immune responses elicited by numerous experimental Envvaccines tested in humans and animals have evoked antibodies that wereunable to neutralize virus or were active against only a very limitedrange of HIV strains (8). Taken together, these findings indicate thatEnv structures found on HIV particles and infected cells can elicitvirus-neutralizing antibodies, whereas candidate vaccines have failed toduplicate or mimic the appropriate Env immunogens needed to evokeefficacious humoral immunity. Moreover, this line of reasoning predictsthat the immune system must be stimulated with authentic Env structuresto elicit antibodies with both Env specificity and virus neutralizingactivity.

Development of a vaccine that will expose the immune system to aproperly configured Env immunogen that is nearly identical to thefunctional structures found on the surface of HIV particles is adifficult technical problem, because the active form of Env is amembrane-bound, unstable, multi-subunit complex. Functional Env proteinis part of trimeric glycoprotein spike that is assembled from subunitsderived from a virus-encoded precursor protein (gp160) (7). Maturationof gp160 involves extensive post-translational modification includingglycosylation, formation of multiple intra-chain disulfide linkages, andproteolytic cleavage into two subunits, which include the smallertransmembrane glycoprotein gp41 and the soluble extracellular subunitgp120. Subunits gp41 and gp120 are held together by noncovalentinteractions to form a monomer and three Env monomers associate into acomplex to form the functional trimeric spikes found on surface of theHIV particles and infected cells. Design and production of a vaccinethat can deliver natively configured trimeric spike immunogens must takeinto account: 1) synthesis of immunogens that are modified extensivelyand correctly; 2) assembly of a stable multisubunit complex heldtogether by labile noncovalent interactions; 3) known insolubility ofthe native gp41 subunit; and 4) the native spike complex is naturallyanchored to the lipid bilayer of cells and virus particles. Thesebiochemical features make it difficult to produce a soluble proteinimmunogen that accurately mimics a trimeric spike; thus, a vectorplatform that will direct synthesis of the native Env spike followingvaccine administration is a more practical approach.

Development of a vector that will produce abundant trimeric Envimmunogen after vaccine administration also is technically challenging.This is due to the fact that vectors encoding a full-length Envpolypeptide tend to be genetically unstable and often express theprotein poorly. This attributed to multiple factors including inhibitoryelements in HIV nucleotide sequences (32), features of the protein thatinhibit efficient translation and post-translational processing (11, 18,37, 39, 40), and the inherent cytotoxicity caused by expression of thistransmembrane glycoprotein. Nucleotide sequence “optimization” methodsand a variety of protein modifications also have been tested withvarying degrees of success including exchange of secretion signals,removal of glycosylation signals, substitution of transmembrane domains,or deletion or replacement of cytoplasmic domain of gp41 (11, 18, 22,37, 39, 40).

The methods used before to improve Env expression are not applicable toall vectors and immunogen designs. For example, protein modificationsintroduced to improve expression might alter structural conformationproducing unpredictable effects on important epitopes. Nucleotidesequence optimization also can have unpredictable effects. Notably,common gene optimization procedures generally use a codon biasreflecting highly expressed mammalian mRNAs, which may not be compatiblewith viral vectors that have genomes with much different nucleotidecompositions. This can be illustrated with the HIV Env gene. Usingonline web tools (28, 30) to design an Env gene with a codon biasreflecting highly expressed mammalian genes (13, 41) results in a codingsequence that has a guanine plus cytosine (G+C) content of approximately60%. Gene inserts with high G+C content like this might be poorlyexpressed or unstable when inserted into vectors like those based on RNAviruses such as measles virus, bovine parainfluenza virus, and vesicularstomatitis virus, which have noticeably lower genomic G+C content of47%, 36%, and 42%, respectively.

Applicants' goal is to make genetically stable vaccine vectors thatabundantly express trimeric membrane-bound Env spikes that closely mimicthe structural properties of the functional glycoprotein complex foundon HIV particles. To achieve this objective, Applicants have developed astrategy to design gene inserts that are tailored to the vector. Toensure that the modified Env immunogens encoded by Applicants' vectorsare processed accurately and retain a native configuration, Applicantshave developed a method to confirm the presence of different classes ofepitopes known to bind with virus-neutralizing antibodies (FIG. 1). Incells expressing the Env immunogens, FACS analysis is used with antibodyprobes including monoclonal antibodies that recognize the CD4 bindingsite (VRC01 and b12) (4, 12, 38), determinant formed by the V1/V2 loops(PG9 and PG16) (36), structures formed by the glycosylated sequences inthe V3 loop (PGT121, PGT126, PGT128, and PGT130) (35), themembrane-proximal external region (2F5 and 4E10) (24, 46), and uniquestructures formed specifically by the trimeric spike (PGT145) (35). Thepresence of epitopes is detected on the cell surface by FACs to confirmthat the vector is directing expression of a correctly processed andassembled Env spike.

Gene Insert Optimization Method Applied to HIV Env.

The foreign gene insert optimization strategy Applicants have developedis illustrated below using HIV Env (strain JR-FL, subtype B). Adaptationof this method to other types of gene inserts and vectors is includedbelow: EnvF fusions proteins for canine distemper virus (CDV) vectors;SIV genes for CDV; SIV genes for VSV; HIV Gag for VSV; HIVCON for CDV.

In summary, the major elements of the Env gene insert optimizationstrategy are:

-   5. Design of the Env gene insert using a nucleotide bias    characteristic of the VSV genome-   6. Removal of RNA sequence elements that might cause VSV genome    instability, inhibit protein translation, reduce mRNA stability, or    promote unwanted RNA processing.-   7. Addition of cis-acting RNA sequences that promote efficient    translation.-   8. Design of a hybrid Env immunogen in which select Env functional    domains are substituted with sequences from the VSV glycoprotein (G)    to promote improved protein expression. Domains from G found to    improve expression while retaining Env structures recognized by    neutralizing antibodies include: a) the VSV G signal sequence    (secretory signal); b) the membrane proximal extracellular stem    domain; c) the transmembrane domain; and/or the intracellular    cytoplasmic domain. It is important to note that in some instances,    replacement of Env domains with sequences from G was not beneficial.    For example, the VSV signal sequence did not enhance expression or    genetic stability in some VSV vectors, but further investigation    revealed that substitution of the signal sequence from the cellular    CD5 protein was beneficial. Thus, the domain swap strategy is    generally applicable, but requires some empirical determination to    achieve the greatest benefit.

HIV-1 Env (Subtype B, strain JR-FL) Gene Insert Design for VSV. Designof HIV-1 Env (subtype B, strain JR-FL) gene inserts for expression fromVSV vectors.

Nucleotide sequence design:

-   -   8. The Optimizer Web Tool (28) was used to generate a new Env        coding sequence with a codon bias similar to VSV.    -   9. The computer-generated sequence was then scanned for        sequences that might have a negative effect on RNA transcription        or stability, translation, or VSV genome replication. These        sequences were removed by replacing nucleotides in the synthetic        Env coding sequence with synonymous codons. Sequences targeted        for substitution included:    -   f. Most homopolymer stretches nucleotides    -   g. Sequences resembling cellular mRNA splicing signals        identified using NNSPLICE webtool (29)    -   h. Sequences resembling known RNA instability elements (43)    -   i. Sequences resembling the known cleavage and polyadenylation        signal (AAUAAA) (42)    -   j. Sequences resembling transcription start and stop signal        consensus for VSV (3)    -   10. The 5′ end of the coding sequences was modified to include a        translation initiation context resembling a consensus for the        other VSV genes (GTCATCATG)    -   11. Stop codon context was modified to resemble highly-expressed        human genes according to Kochetov (13)    -   12. 5′ end includes a HindIII site for cloning into the rVSV        genomic cDNA.    -   13. For molecular cloning purposes, two sequences were edited to        remove HindIII sites in the coding sequence.    -   14. 3′ end was modified by addition of a BamHI site for cloning        into the rVSV genomic cDNA

Protein Modifications. Multiple domain swaps (FIG. 2) have been testedidentifying several that 1) improve Env expression; 2) retain functionalproperties of native Env; and 3) preserve critical structuraldeterminants recognized by neutralizing antibodies.

TABLE 1 Amino acid coordinates for sequences of HIV Env and VSV Gincorporated into EnvG hybrids illustrated in FIG. 2. N-Terminal SignalProtein Name Peptide Env (JR-FL) VSV G A VSV G 1-17 VSV G None  18-511 BEnv (JR-FL) 1-29 Env 30-847 None C EnvG-CT 1-17 VSV 30-695 483-511 DEnvG-CTR * 1-17 VSV 30-700 486-511 E EnvG-TM 1-17 VSV 30-674 463-511 FEnvG-SS 1-17 30-650 446-511 G EnvG-LS 1-17 30-650 426-511 H EnvG-XLS1-17 30-650 395-511

HIV-1 Env (Subtype A, strain BG505) Gene Insert Design for VSV. Asynthetic gene sequence encoding HIV-1 Env (subtype A, strain BG505) wasdesigned for VSV vectors as described above with some modifications.

Nucleotide sequence design:

-   -   13. The Optimizer webtool (28) was used to generate a new BG505        Env coding sequence that has a codon bias similar to VSV.    -   14. Homopolymer sequences >5 nucleotides (CCCCC, GGGGG) were        interrupted by substitution of at least one synonymous codon.    -   15. Homopolymer sequences >4 nculeotides (AAAA, TTTT) were        interrupted by substitution with at least one synonymous codon.    -   16. The 5′ end of the coding sequences was modified to include a        Kozak translation initiation sequence (aggaGCCACCATG (SEQ ID NO:        1)) (16)    -   17. An optimal translation termination signal TAAag was added to        the 3′ end of the coding sequences (13)    -   18. RNA instability elements similar to UUAUUUAUU (43) were        interrupted by replacing sequence with synonymous codons.    -   19. Potential polyadenylation signals (AAUAAA) (42) were        interrupted by substitution with synonymous codons.    -   20. Potential T7 polymerase terminators were removed: cTGAg,        gacTAAag, ctTAAac and gacTAAat to prevent inhibition of        recombinant VSV rescue from cloned DNA (21)    -   21. The mRNA splice site prediction tool NNSPLICE webtool (29)        was used to predict the potential splice sites and synonymous        codons were substituted.    -   22. A NheI(GCTAGC) site was added to the 3′ end of VSV-G signal        peptide    -   23. 5′ end was modified to include a BstBI site for cloning into        the rVSV genomic cDNA.    -   24. 3′ end was modified to include a PacI site for cloning into        the rVSV genomic cDNA

Protein modifications: In this example, an EnvG hybrid was designed withthe VSV G signal peptide, transmembrane and cytoplasmic domains.

HIV-1 Env (Subtype C, 16055) Gene Insert Design for VSV. Nucleotidesequence design: A synthetic HIV Env gene (subtype C, strain 16055) wasdesigned for incorporation into VSV vectors using the steps describedabove.

Protein domain modifications: In this example, an EnvG hybrid wasdesigned with the cellular CD5 signal peptide and the VSV Gtransmembrane and cytoplasmic domains.

HIV-1 Env (Subtype B, Strain JR-FL) Gene Insert Design for VSV EnablingStable Coexpression of Env and VSV G.

Nucleotide sequence design. A synthetic HIV Env gene (subtype B, strainJR-FL) was designed for incorporation into VSV vectors using the stepsdescribed in 2.2.1 with some modification. The EnvG hybrid was designedspecifically for making stable VSV vectors that coexpress VSV G and Env.

-   -   12. The Optimizer webtool (28) was used to generate a new strain        JR-FL Env coding sequence that has a codon bias similar to VSV.    -   13. Homopolymer sequences >5 nucleotides (CCCCC, GGGGG) were        interrupted by substitution of at least one synonymous codon.    -   14. Homopolymer sequences >4 nucleotides (AAAA, TTTT) were        interrupted by substitution with at least one synonymous codon.    -   15. The 5′ end of the coding sequences was modified to include a        Kozak translation initiation sequence (aggaGCCACCATG (SEQ ID NO:        1)) (16)    -   16. An optimal translation termination signal TGAg was added to        the 3′ end of the coding sequences (13)    -   17. RNA instability elements similar to UUAUUUAUU (43) were        interrupted by replacing sequence with synonymous codons.    -   18. Potential polyadenylation signals (AAUAAA) (42) were        interrupted by substitution with synonymous codons.    -   19. Potential T7 polymerase terminators were removed: cTGAg,        gacTAAag, ctTAAac and gacTAAat to prevent inhibition of        recombinant VSV rescue from cloned DNA (21)    -   20. The mRNA splice site prediction tool NNSPLICE webtool (29)        was used to predict the potential splice sites and synonymous        codons were substituted.    -   21. 5′ end was modified to include a BstBI site for cloning into        the rVSV genomic cDNA.    -   22. 3′ end was modified to include a PacI site for cloning into        the rVSV genomic cDNA

Protein domain modifications: In this example, an EnvG hybrid wasdesigned with the cellular CD5 signal peptide and the VSV Gtransmembrane and cytoplasmic domains. Additionally, an Alanine residuewas added at the C-terminus of the signal peptide to improve agreementwith signal peptidase cleavage signals (online webtool (27)).

Expression of the EnvG hybrid from a VSV vector is illustrated in theWestern blot in FIG. 6. Infected Vero cell lysate and purified virusparticles were analyzed by SDS polyacrylamide gel electrophoresis andWestern blot analysis. The Western blot was probed with monoclonalantibody 2F5, which recognizes two forms of Env protein; the gp160precursor and the gp41 subunit produced by proteolytic cleavage.

Example 3 Application of the Gene Design Strategy to Other Vectors andGene Inserts

It is important to note that Applicants' gene insert optimizationapproach is not restricted to VSV vectors and the strategy can bemodified for use across multiple vector platforms. Examples of Env geneinserts designed for expression from canine distemper virus (CDV)vectors are provided below. Env gene inserts with nucleotide sequenceresembling the CDV genome can be produced and functional domains fromthe CDV fusion (F) protein can be functionally substituted into Env asdescribed above for VSV G.

The gene optimization procedure also is not limited to Env immunogens.Changes to nucleotide sequence to reflect the base composition of theviral vector genome can improve the stability of other inserts. Inaddition, vector stability can be improved by altering specific proteinsignals that modulate export, posttranslational modification, orcellular localization. Examples below include an HIV gag gene insertdesigned for expression from VSV, SIV Gag and Env genes designed forexpression by CDV, and SIV Gag and Env genes designed for expressionfrom VSV, and a fusion protein called the HIVCON designed for expressionby CDV.

Finally, Applicants have found that gene insert designed as describedabove also can be used in plasmid DNA expression vectors. For example,genes designed for VSV also expressed efficiently from plasmid DNAvectors. This result indicates that elements of the gene design approachApplicants have developed have more broad applicability.

HIV Env Gene Designs for Expression from CDV Vectors.

Synthetic genes encoding HIV Env were designed for expression from avector developed from an attenuated strain of canine distemper virus(CDV). Vectors expressing HIV Env subtype A (BG505) and subtype C(16055) Env have been isolated. Plaques formed by infection of Vero cellmonolayers are shown in FIG. 7.

Nucleotide Sequence Design:

A synthetic HIV Env gene (subtype A, BG505) was designed forincorporation into CDV vectors steps similar to those described abovewith modifications. Importantly, the synthetic gene was designed using acodon bias characteristic of the CDV genome.

The gene was designed using the following steps:

-   -   9. The Optimizer Web Tool (28) was used to generate a synthetic        Env gene with a nucleotide content similar to the CDV genome.        CDV codon preference was retrieved from database (2,5).    -   10. Homopolymer sequences >5 nucleotides were interrupted by        substitution of at least one synonymous codon.    -   11. The 5′ end of the coding sequences was modified to include a        Kozak translation initiation sequence (aggaGCCACCATG (SEQ ID NO:        1)) (16)    -   12. RNA instability elements similar to UUAUUUAUU (43) were        interrupted by replacing sequence with synonymous codons.    -   13. Potential polyadenylation signals (AAUAAA) (42) were        interrupted by substitution with synonymous codons.    -   14. Potential T7 polymerase terminators were removed to prevent        inhibition of recombinant CDV rescue from cloned DNA (21).    -   15. The mRNA splice site prediction tool webtool NNSPLICE (29)        was used to predict the potential splice sites and synonymous        codons were substituted.    -   16. Adjust insert length to ensure that introduction into the        CDV genome would follow the ‘Rule-of-Six” (i.e. total CDV genome        length is multiples of 6) (14).

Protein domain modifications: In this example, the Env genes weredesigned to encode a truncated Env proteins that lack the cytoplasmictail.

SIV Env gene designed for expression from VSV. Nucleotide sequencedesign: An SIV (strain SIVmac239) Env gene was designed for expressionby VSV vectors. As described below:

-   -   12. Replace the SIV signal sequence with VSV signal sequence,        keeping the first amino acid after cleavage on SIV Env.    -   13. Replace the SIV Env cytoplasmic tail with VSV G.    -   14. The Optimizer Web Tool (28) was used to generate synthetic        coding sequence with a codon bias similar to VSV based on the        codon usage for Vesicular Stomatitis Indiana Virus (25).    -   15. Most homopolymer stretches nucleotides were interrupted by        substitution of one or more synonymous codon(s), using the VSV        codon usage table to maintain codon bias similar to VSV.    -   16. The 5′ end of the coding sequences was modified to include a        Kozak translation initiation sequence (aggaGCCACCATG) (16)    -   17. An optimal translation termination signal TAAag was added to        the 3′ end of the coding sequences (13)    -   18. The mRNA splice site prediction tool NNSPLICE webtool (29)        was used to predict the potential splice sites, which were        substituted with synonymous codons.    -   19. RNA instability elements similar to UUAUUUAUU (43) were        interrupted by replacing sequence with synonymous codons.    -   20. Potential polyadenylation signals (AAUAAA) (42) were        interrupted by substitution with synonymous codons.    -   21. Potential T7 polymerase terminators were removed: cTGAg,        gacTAAag, ctTAAac and gacTAAat to prevent inhibition of        recombinant VSV rescue from cloned DNA (21)

Protein modifications: The C-terminal cytoplasmic domain was removedfrom SIV Env.

HIVCON Gene Design for Expression from CDV.

The HIVCON is an HIV immunogen designed by Letourneau et al (17). Theimmunogen is a fusion protein composed of relatively short, highlyconserved sequence elements from the HIV proteome, which is intended toelicit cellular immunity against targets that cannot be easily mutatedby the virus. The HIVCON insert designed by Hanke et al was designedspecifically for expression by plasmid DNA and DNA virus vectors.Applicants have made several new gene inserts designed for incorporationinto canine distemper virus (CDV) vectors, which have a smallnegative-sense, single-stranded RNA genome.

To design a gene that was more compatible with a CDV vector, a syntheticHIVCON coding sequence (corresponding to FIG. 8C) was prepared using thefollowing steps:

Nucleotide sequence design:

-   -   10. The optimizer Web Tool (28) was used to generate an HIVCON        coding sequence that has a codon bias similar to CDV based on        the Codon Usage for Canine Distemper Virus (25).    -   11. All homopolymer stretches >4-5 nucleotides were interrupted        using an alternative codon from the CDV codon usage table to        maintain codon bias similar to CDV.    -   12. All sequences that resemble morbillivirus transcription stop        or start signals (26, 33) were interrupted using an alternative        codon from the CDV codon usage table.    -   13. Potential RNA splicing signals were identified using the        NNSPLICE webtool (29) and subsequently interrupted by        substitution with synonymous codons    -   14. RNA instability elements similar to 5-UUAUUUAUU-3′ (43) were        interrupted by silent mutation using an alternative codon from        the CDV codon usage table to maintain codon bias similar to CDV.    -   15. Two sequences resembling T7 RNA polymerase terminators were        interrupted using synonymous codons (21).    -   16. A Kozak sequence GGAGCCACC was inserted to improve        translation (15).    -   17. To increase the stability of the vector and the integrity of        the insert, a 2A-like peptide motif from Thosea asigna virus        (EGRGSLLTCGDVEENPGP (SEQ ID NO: 2)) (20) was added to fuse        HIVCON coding sequence with the with CDV (M) gene.    -   18. Coding sequence for the VSV G leader (MKCLLYLAFLFIGVNCK(SEQ        ID NO: 3)) was also incorporated at the N-terminus to promote        secretion of the polypeptide immunogen.

Protein modifications: The VSV signal peptide was added to theN-terminus. At the C-terminus, a 2A-like polypeptide cleavage signal(20) was added between the HIVCON and matrix coding sequences. Thiscreates a polycistronic HIVCON-Matrix gene that encodes two polypeptidesas depicted in FIG. 9.

Expression of EnvG and EnvF from plasmid DNA vectors. To demonstratethat genes designed by Applicants' approach were not restricted to usein RNA virus vectors like CDV and VSV, Applicants developed plasmidvectors encoding the hybrid the EnvG protein described earlier (CladeA). In addition, using the basic approach described above, Applicantsalso designed hybrid Env proteins (EnvF) in which domains of Env werereplaced with sequences from the CDV fusion (F) protein (9). EnvG andEnvF genes were cloned into plasmid DNA vectors under the control of theunder the control of the cytomegalovirus promoter and enhancer (23).Flow cytometry (EnvG) or Western blotting (EnvF) was used to evaluatetransient expression of Env protein.

Transient Expression of EnvG Hybrids from Plasmid DNA.

Plasmid DNA containing the EnvG (subtype A) described above wastransfected into 293T cells. Cell surface expression of the EnvGproteins and its antigenic profile are illustrated in the cell sortinganalysis shown in FIG. 10.

FIG. 13 depicts transient expression of EnvF hybrids from plasmid DNA.

Four chimeric EnvF constructs (Subtype C, strain 16055) were designedand cloned under CMV promoter in plasmid DNA vector (FIG. 11). Inaddition to coding sequence design described above, F domainsubstitutions were made as illustrated in FIG. 11.

Expression of EnvF proteins was evaluated after transient expression in293T cells. Western blot analysis demonstrated that all four constructswere expressed from transfected plasmids (FIG. 12). The four EnvF fusionprotein also were analyzed to determine whether they retained fusionfunction of the natural HIV Env protein. EnvF2 and EnvF4 were positivefor cell membrane fusion indicating that the domain substitutions didnot abolish this function (data not shown). All four EnvF proteins werepositive for binding to monoclonal antibodies PG9, PG16, B6, B12, andVRC01 when expressed on the cell surface following transient expression(data not shown).

REFERENCES

-   1. Andre, S., B. Seed, J. Eberle, W. Schraut, A. Bultmann, and J.    Haas. 1998. Increased immune response elicited by DNA vaccination    with a synthetic gp120 sequence with optimized codon usage. J Virol    72:1497-1503.-   2. Barouch, D. H. 2006. Rational design of gene-based vaccines. The    Journal of pathology 208:283-289.-   3. Barr, J. N., S. P. Whelan, and G. W. Wertz. 2002. Transcriptional    control of the RNA-dependent RNA polymerase of vesicular stomatitis    virus. Biochim Biophys Acta 1577:337-353.-   4. Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B.    Thornton, P. W. Parren, L. S. Sawyer, R. M. Hendry, N. Dunlop, P. L.    Nara, and et al. 1994. Efficient neutralization of primary isolates    of HIV-1 by a recombinant human monoclonal antibody. Science    266:1024-1027.-   5. Donnelly, J. J., J. B. Ulmer, J. W. Shiver, and M. A. Liu. 1997.    DNA vaccines. Annu Rev Immunol 15:617-648.-   6. Doria-Rose, N. A., R. M. Klein, M. M. Manion, S. O'Dell, A.    Phogat, B. Chakrabarti, C. W. Hallahan, S. A. Migueles, J.    Wrammert, R. Ahmed, M. Nason, R. T. Wyatt, J. R. Mascola, and M.    Connors. 2009. Frequency and phenotype of human immunodeficiency    virus envelope-specific B cells from patients with broadly    cross-neutralizing antibodies. J Virol 83:188-199.-   Freed, E. O., and M. A. Martin. 2007. HIVs and thier replication, p.    2107-2186. In D. M. Knipe, D. E. Griffin, R. A. Lamb, S. E.    Straus, P. M. Howley, M. A. Martin, B. Roizman, and S. E. Straus    (ed.), Fields Virology. Lippincott Williams & Wilkins.-   8. Girard, M. P., and W. C. Koff. 2008. Human immunodeficiency virus    vaccines, p. 1213-1252. In S. A. Plotkin, W. A. Orenstein, and P. A.    Offit (ed.), Vaccines, 5 ed. Elsevier, Philadelphia.-   9. Griffin, D. E. 2007. Measles virus, p. 1551-1586. In D. M.    Knipe, D. E. Griffin, R. A. Lamb, S. E. Straus, P. M. Howley, M. A.    Martin, B. Roizman, and S. E. Straus (ed.), Fields Virology, vol. 2.    Wolters Kluwer, Philadelphia.-   10. Haas, J., E. C. Park, and B. Seed. 1996. Codon usage limitation    in the expression of HIV-1 envelope glycoprotein. Current biology:    CB 6:315-324.-   11. Johnson, J. E., W. Rodgers, and J. K. Rose. 1998. A plasma    membrane localization signal in the HIV-1 envelope cytoplasmic    domain prevents localization at sites of vesicular stomatitis virus    budding and incorporation into VSV virions. Virology 251:244-252.-   12. Kessler, J. A., 2nd, P. M. McKenna, E. A. Emini, C. P.    Chan, M. D. Patel, S. K. Gupta, G. E. Mark, 3rd, C. F. Barbas,    3rd, D. R. Burton, and A. J. Conley. 1997. Recombinant human    monoclonal antibody IgG1b12 neutralizes diverse human    immunodeficiency virus type 1 primary isolates. AIDS research and    human retroviruses 13:575-582.-   13. Kochetov, A. V., I. V. Ischenko, D. G. Vorobiev, A. E.    Kel, V. N. Babenko, L. L. Kisselev, and N. A. Kolchanov. 1998.    Eukaryotic mRNAs encoding abundant and scarce proteins are    statistically dissimilar in many structural features. FEBS letters    440:351-355.-   14. Kolakofsky, D., T. Pelet, D. Garcin, S. Hausmann, J. Curran,    and L. Roux. 1998. Paramyxovirus RNA synthesis and the requirement    for hexamer genome length: the rule of six revisited. J Virol    72:891-899.-   15. Kozak, M. 1999. Initiation of translation in prokaryotes and    eukaryotes. Gene 234:187-208.-   16. Kozak, M. 1991. Structural features in eukaryotic mRNAs that    modulate the initiation of translation. J Biol Chem 266:19867-19870.-   17. Letourneau, S., E. J. Im, T. Mashishi, C. Brereton, A.    Bridgeman, H. Yang, L. Dorrell, T. Dong, B. Korber, A. J. McMichael,    and T. Hanke. 2007. Design and pre-clinical evaluation of a    universal HIV-1 vaccine. PLoS ONE 2:e984.-   18. Li, Y., L. Luo, D. Y. Thomas, and C. Y. Kang. 1994. Control of    expression, glycosylation, and secretion of HIV-1 gp120 by    homologous and heterologous signal sequences. Virology 204:266-278.-   19. Li, Y., K. Svehla, M. K. Louder, D. Wycuff, S. Phogat, M.    Tang, S. A. Migueles, X. Wu, A. Phogat, G. M. Shaw, M. Connors, J.    Hoxie, J. R. Mascola, and R. Wyatt. 2009. Analysis of neutralization    specificities in polyclonal sera derived from human immunodeficiency    virus type 1-infected individuals. J Virol 83:1045-1059. 20.    Luke, G. A., P. de Felipe, A. Lukashev, S. E. Kallioinen, E. A.    Bruno, and M. D. Ryan. 2008. Occurrence, function and evolutionary    origins of ‘2A-like’ sequences in virus genomes. J Gen Virol    89:1036-1042.-   21. Macdonald, L. E., R. K. Durbin, J. J. Dunn, and W. T.    McAllister. 1994. Characterization of two types of termination    signal for bacteriophage T7 RNA polymerase. Journal of molecular    biology 238:145-158.-   22. Megati, S., D. Garcia-Hand, S. Cappello, V. Roopchand, A.    Masood, R. Xu, A. Luckay, S. Y. Chong, M. Rosati, S. Sackitey, D. B.    Weiner, B. K. Felber, G. N. Pavlakis, Z. R. Israel, L. R.    Smith, J. H. Eldridge, M. K. Sidhu, and M. A. Egan. 2008. Modifying    the HIV-1 env gp160 gene to improve pDNA vaccine-elicited    cell-mediated immune responses. Vaccine 26:5083-5094.-   23. Meier, J. L., and M. F. Stinski. 1996. Regulation of human    cytomegalovirus immediate-early gene expression. Intervirology    39:331-342.-   24. Muster, T., F. Steindl, M. Purtscher, A. Trkola, A. Klima, G.    Himmler, F. Ruker, and H. Katinger. 1993. A conserved neutralizing    epitope on gp41 of human immunodeficiency virus type 1. Journal of    virology 67:6642-6647.-   25. Nakamura, Y., T. Gojobori, and T. Ikemura. 2000. Codon usage    tabulated from international DNA sequence databases: status for the    year 2000. Nucleic Acids Res 28:292.-   26. Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu,    and S. A. Udem. 2001. Analysis of the noncoding regions of measles    virus strains in the Edmonston vaccine lineage. J Virol 75:921-933.-   27. Petersen, T. N., S. Brunak, G. von Heijne, and H. Nielsen. 2011.    SignalP 4.0: discriminating signal peptides from transmembrane    regions. Nature methods 8:785-786.-   28. Puigbo, P., E. Guzman, A. Romeu, and S. Garcia-Vallve. 2007.    OPTIMIZER: a web server for optimizing the codon usage of DNA    sequences. Nucleic Acids Res 35:W 126-131-   29. Reese, M. G., F. H. Eeckman, D. Kulp, and D. Haussler. 1997.    Improved splice site detection in Genie. J Comput Biol 4:311-323.-   30. Richardson, S. M., P. W. Nunley, R. M. Yarrington, J. D. Boeke,    and J. S. Bader. 2010. GeneDesign 3.0 is an updated synthetic    biology toolkit. Nucleic Acids Res 38:2603-2606.-   31. Sather, D. N., J. Armann, L. K. Ching, A. Mavrantoni, G.    Sellhorn, Z. Caldwell, X. Yu, B. Wood, S. Self, S. Kalams, and L.    Stamatatos. 2009. Factors associated with the development of    cross-reactive neutralizing antibodies during human immunodeficiency    virus type 1 infection. J Virol 83:757-769.-   32. Schwartz, S., M. Campbell, G. Nasioulas, J. Harrison, B. K.    Felber, and G. N. Pavlakis. 1992. Mutational inactivation of an    inhibitory sequence in human immunodeficiency virus type 1 results    in Rev-independent gag expression. J Virol 66:7176-7182.-   33. Sidhu, M. S., W. Husar, S. D. Cook, P. C. Dowling, and S. A.    Udem. 1993. Canine distemper terminal and intergenic non-protein    coding nucleotide sequences: completion of the entire CDV genome    sequence. Virology 193:66-72.-   34. Stamatatos, L., L. Morris, D. R. Burton, and J. R.    Mascola. 2009. Neutralizing antibodies generated during natural    HIV-1 infection: good news for an HIV-1 vaccine? Nature medicine    15:866-870.-   35. Walker, L. M., M. Huber, K. J. Doores, E. Falkowska, R.    Pejchal, J. P. Julien, S. K. Wang, A. Ramos, P. Y. Chan-Hui, M.    Moyle, J. L. Mitcham, P. W. Hammond, 0. A. Olsen, P. Phung, S.    Fling, C. H. Wong, S. Phogat, T. Wrin, M. D. Simek, W. C.    Koff, I. A. Wilson, D. R. Burton, and P. Poignard. 2011. Broad    neutralization coverage of HIV by multiple highly potent antibodies.    Nature 477:466-470.-   36. Walker, L. M., S. K. Phogat, P. Y. Chan-Hui, D. Wagner, P.    Phung, J. L. Goss, T. Wrin, M. D. Simek, S. Fling, J. L.    Mitcham, J. K. Lehrman, F. H. Priddy, O. A. Olsen, S. M. Frey, P. W.    Hammond, S. Kaminsky, T. Zamb, M. Moyle, W. C. Koff, P. Poignard,    and D. R. Burton. 2009. Broad and potent neutralizing antibodies    from an African donor reveal a new HIV-1 vaccine target. Science    326:285-289.-   37. Wang, B. Z., W. Liu, S. M. Kang, M. Alam, C. Huang, L. Ye, Y.    Sun, Y. Li, D. L. Kothe, P. Pushko, T. Dokland, B. F. Haynes, G.    Smith, B. H. Hahn, and R. W. Compans. 2007. Incorporation of high    levels of chimeric human immunodeficiency virus envelope    glycoproteins into virus-like particles. J Virol 81:10869-10878.-   38. Wu, X., Z. Y. Yang, Y. Li, C. M. Hogerkorp, W. R. Schief, M. S.    Seaman, T. Zhou, S. D. Schmidt, L. Wu, L. Xu, N. S. Longo, K.    McKee, S. O'Dell, M. K. Louder, D. L. Wycuff, Y. Feng, M. Nason, N.    Doria-Rose, M. Connors, P. D. Kwong, M. Roederer, R. T. Wyatt, G. J.    Nabel, and J. R. Mascola. 2010. Rational design of envelope    identifies broadly neutralizing human monoclonal antibodies to    HIV-1. Science 329:856-861-   39. Wyatt, L. S., I. M. Belyakov, P. L. Earl, J. A. Berzofsky,    and B. Moss. 2008. Enhanced cell surface expression, immunogenicity    and genetic stability resulting from a spontaneous truncation of HIV    Env expressed by a recombinant MVA. Virology 372:260-272.-   40. Wyatt, L. S., P. L. Earl, W. Xiao, J. L. Americo, C. A.    Cotter, J. Vogt, and B. Moss. 2009. Elucidating and minimizing the    loss by recombinant vaccinia virus of human immunodeficiency virus    gene expression resulting from spontaneous mutations and positive    selection. J Virol 83:7176-7184.-   41. Zhang, M. Q. 1998. Statistical features of human exons and their    flanking regions. Human molecular genetics 7:919-932.-   42. Zhao, J., L. Hyman, and C. Moore. 1999. Formation of mRNA 3′    ends in eukaryotes: mechanism, regulation, and interrelationships    with other steps in mRNA synthesis. Microbiology and molecular    biology reviews: MMBR 63:405-445.-   43. Zubiaga, A. M., J. G. Belasco, and M. E. Greenberg. 1995. The    nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates    mRNA degradation. Mol Cell Biol 15:2219-2230.-   44. Zwick, M. B., L. L. Bonnycastle, A. Menendez, M. B.    Irving, C. F. Barbas, 3rd, P. W. Parren, D. R. Burton, and J. K.    Scott. 2001. Identification and characterization of a peptide that    specifically binds the human, broadly neutralizing anti-human    immunodeficiency virus type 1 antibody b12. J Virol 75:6692-6699.-   45. Zwick, M. B., and D. R. Burton. 2007. HIV-1 neutralization:    mechanisms and relevance to vaccine design. Curr HIV Res 5:608-624.-   46. Zwick, M. B., A. F. Labrijn, M. Wang, C. Spenlehauer, E. O.    Saphire, J. M. Binley, J. P. Moore, G. Stiegler, H. Katinger, D. R.    Burton, and P. W. Parren. 2001. Broadly neutralizing antibodies    targeted to the membrane-proximal external region of human    immunodeficiency virus type 1 glycoprotein gp41. J Virol    75:10892-10905.

The invention is further described by the following numbered paragraphs:

1. A method for developing a gene insert compatible with a host vectorcomprising:

-   -   a. modifying a protein sequence of the gene insert to lessen        potential interference with vector propagation while ensuring        that the protein encoded by the gene insert is expressed and        processed efficiently and maintains desired structural features        and    -   b. designing the gene insert with a nucleotide sequence that        resembles a base composition of the host vector.

2. The method of paragraph 1, wherein the gene insert is designed with acodon bias similar to the host vector.

3. The method of paragraph 1 or 2, wherein the nucleotide sequenceelements in the gene insert resemble known hot spots for mutation,inhibitors of replication or gene expression or direct inappropriate RNAprocessing are interrupted with synonymous codons.

4. The method of any one of paragraphs 1-3, wherein the desiredstructural features that modulate translation, post-translationalprocessing and cellular compartmentalization are abundantly expressedand have a less negative effect on vector propagation and fitness.

5. The method of any one of paragraphs 1-4, wherein the gene insert isHIV Env.

6. The method of paragraph 5, wherein the host vector is VSV.

7. The method of paragraph 6, wherein the Env gene insert is designedwith a nucleotide bias characteristic of VSV.

8. The method of paragraph 6 or 7, wherein RNA sequence elements thatmight cause VSV genome instability, inhibit protein translation, reducemRNA stability, or promote unwanted RNA processing are removed.

9. The method of any one of paragraphs 6-8, wherein cis-acting RNAsequences that promote efficient translation are added.

10. The method of any one of paragraphs 6-9, wherein the Env gene insertis substituted with sequences from VSV glycoprotein (G).

11. The method of paragraph 10 wherein the sequences from VSV G areselected from the group consisting of the VSV G signal sequence(secretory signal); the membrane proximal extracellular stem domain; thetransmembrane domain; and/or the intracellular cytoplasmic domain.

12. The method of any one of paragraphs 6-11, wherein the codon bias ofthe Env gene insert is similar to VSV.

13. The method of paragraph 12, wherein the codon bias is changed to asequence consisting of homopolymer stretches >5 nucleotides, sequencesresembling cellular mRNA splicing signals, sequences resembling RNAinstability elements, sequences resembling a cleavage andpolyadenylation signal (AAUAAA) and sequences resembling transcriptionstart and stop signal consensus for VSV.

14. The method of any one of paragraphs 1-5, wherein the host vector iscanine distemper virus (CDV).

15. The method of paragraph 14, wherein functional domains from the CDVfusion (F) protein are substituted into Env gene insert.

16. The method of any one of paragraphs 1-4, wherein the gene insert isHIV gag, SIV gag, SIV Env, or HIVCON.

17. The method of paragraph 16, wherein the host vector is CDV, VSV or aplasmid vector.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A method for improving protein expression, vectorpropagation, and genetic stability in a virus host vector selected fromthe group consisting of vesicular stomatitis virus (VSV) and caninedistemper virus (CDV) comprising: (a) developing a gene insertcompatible with the virus host vector comprising substituting synonymouscodons in the nucleotide sequence of the gene insert, wherein the codonsare selected from the codons used at the highest frequency for the virushost vector; (b) cloning the gene insert into the virus host vector,thereby improving protein expression, vector propagation, and geneticstability in the virus host vector; and (c) expressing the proteinencoded by the vector in a cell.
 2. The method of claim 1, wherein thevirus is VSV and the G+C content of the nucleotide sequence of the geneinsert is between 40% and 42%.
 3. The method of claim 1, wherein thevirus is CDV and the G+C content of the nucleotide sequence is between32% and 33%.
 4. The method of claim 1, wherein the gene insert isselected from the group consisting of human immunodeficiency virus (HIV)envelope glycoprotein (Env) and simian immunodeficiency virus (SIV) Env.5. The method of claim 4, wherein the Env gene insert is a hybrid thatincludes sequences encoding functional domains substituted withsequences encoding analogous functional domains from VSV glycoprotein(G) selected from the group consisting of the VSV G signal sequence; themembrane proximal extracellular stem domain; the transmembrane domain;and/or the intracellular cytoplasmic domain.
 6. The method of claim 5,wherein the Env signal sequence, transmembrane domain, and intracellularcytoplasmic domain are substituted with the VSV G signal sequence,transmembrane domain, and intracellular cytoplasmic domain.
 7. Themethod of claim 1, wherein cis-acting RNA sequences for enhancingtranslation are added and wherein translation is enhanced in comparisonto a vector without the sequences.
 8. The method of claim 1, furthercomprising one or more modifications to the nucleotide sequence of thegene insert comprising: interrupting homopolymer sequences >5nucleotides CCCCC and GGGGG by substitution of at least one synonymouscodon; interrupting homopolymer sequences >4 nucleotides AAAA and TTTTwith at least one synonymous codon; modifying the 5′ end of the codingsequences to include a Kozak translation initiation sequenceaggaGCCACCATG (SEQ ID NO:1); adding an optimal translation terminationsignal TAAag to the 3′ end of the coding sequences; interrupting RNAinstability elements comprising UUAUUUAUU by replacement with synonymouscodons; interrupting a potential polyadenylation signal comprisingAAUAAA by substitution with synonymous codons; or removing potential T7RNA polymerase terminators cTGAg, gacTAAag, ctTAAac and gacTAAat toprevent inhibition of recombinant VSV rescue from cloned DNA.
 9. Themethod of claim 1, further comprising measuring binding of the proteinto antibodies specific to the gene insert.
 10. A method for improvingprotein expression of an immunodeficiency virus (HIV) envelopeglycoprotein (Env) gene insert in a vesicular stomatitis virus (VSV)host vector comprising: (a) substituting the Env signal sequence,transmembrane domain, and intracellular cytoplasmic domain with the VSVG signal sequence, transmembrane domain, and intracellular cytoplasmicdomain; (b) cloning the gene insert into the virus host vector, therebyimproving protein expression in the host vector; and (c) expressing theprotein encoded by the vector in a cell.
 11. The method of claim 10,further comprising substituting synonymous codons in the nucleotidesequence of the gene insert, wherein the codons are selected from thecodons used at the highest frequency for VSV.
 12. The method of claim11, wherein the G+C content of the nucleotide sequence of the geneinsert is between 40% and 42%.
 13. The method of claim 10, furthercomprising one or more modifications to the nucleotide sequence of thegene insert comprising: interrupting homopolymer sequences >5nucleotides CCCCC and GGGGG by substitution of at least one synonymouscodon; interrupting homopolymer sequences >4 nucleotides AAAA and TTTTwith at least one synonymous codon; modifying the 5′ end of the codingsequences to include a Kozak translation initiation sequenceaggaGCCACCATG (SEQ ID NO:1); adding an optimal translation terminationsignal TAAag to the 3′ end of the coding sequences; interrupting RNAinstability elements comprising UUAUUUAUU by replacement with synonymouscodons; interrupting a potential polyadenylation signal comprisingAAUAAA by substitution with synonymous codons; or removing potential T7RNA polymerase terminators cTGAg, gacTAAag, ctTAAac and gacTAAat toprevent inhibition of recombinant VSV rescue from cloned DNA.
 14. Themethod of claim 10, wherein cis-acting RNA sequences for enhancingtranslation are added and wherein translation is enhanced in comparisonto a vector without the sequences.
 15. The method of claim 10, furthercomprising measuring binding of the protein to antibodies specific tothe gene insert.